Formulation of a peptide vaccine

ABSTRACT

The invention relates to a novel reconstitution composition, a pharmaceutical composition and kit of parts comprising said reconstitution composition. The invention further relates to a method of treatment using said pharmaceutical composition and/or the pharmaceutical composition for use as a medicament. Also provided is a method for reconstituting dried peptides and a method for preparing a pharmaceutical composition using the reconstitution composition of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/311,629, filed Dec. 19, 2018, which is the National Phase ofInternational Patent Application No. PCT/EP2017/064882, filed Jun. 19,2017, published as WO 2017/220463, which claims priority to EuropeanApplication No. 16175215.9, filed Jun. 20, 2016. The contents of theseapplications are herein incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 15, 2018, isnamed SequenceListing.txt and is 109,817 bytes.

FIELD OF THE INVENTION

This invention is in the field of medicine and immunology. Inparticular, it relates to a novel composition for reconstitutingpeptides for vaccination. This composition is in particular suitable forpreparing pharmaceutical peptide-based vaccines that further comprise anoil-based adjuvant.

BACKGROUND OF THE INVENTION

Clinical results have indicated that the era of successful therapeuticvaccination has arrived. Regression of lesions was shown forpremalignant lesions caused by HPV and the clinical benefit of prolongedsurvival has been established. Vaccines based on synthetic long peptidesare among the optimal vaccine platforms. Peptide vaccine orpeptides-based vaccines are developed for the treatment of persistentinfections and cancer, preferably targeting the immune system to clearcells that express viral antigens, cancer-antigens and/or neo-antigens.It is appreciated in the art that peptide-based vaccines capable ofeliciting an effective cellular immune response (CD4⁺ and CD8⁺ T-cellresponse) targeting antigen-specific cytotoxic T cells capable ofclearing the antigen-expressing cells. Antigens of choice include mutantsequences, selected cancer testis antigens and viral antigens (forreview, see Melief et al. 2016 Journal of Clinical Investigation, Vol125(9) pages 3401-3412).

One of the challenges of peptide-based vaccines is to provide forphysically and chemically stable injectable solutions. This is inparticular a challenge for peptide-based vaccine emulsions comprisingmore than one peptide and oil-based adjuvants. Injectable vaccinesolutions are typically prepared on-site about 1 to 3 hours beforeadministration to the patient using dried, mostly lyophilized peptidesas a starting material. Therefore, there is a need for a suitablereconstitution composition that allows for the fast reconstitution ofdried peptides, which can subsequently be admixed easily with oil-basedadjuvants resulting in an emulsion that is physically and chemicallystable for at least 2 to 3 hours storage at room temperature beforebeing administered to the patient.

DETAILED DESCRIPTION OF THE INVENTION Reconstitution Composition

Provided is a novel composition for reconstituting peptides forvaccination. This reconstitution composition comprises or consists ofabout 60-80% v/v aqueous solution comprising an organic acid, about5-10% v/v propylene glycol (CAS no. 57-55-6), about 10-20% v/v loweralcohol and about 5-10% v/v non-ionic hydrophilic surfactant.

Preferably, the organic acid is a weak organic acid such as a carboxylicacid. A weak organic acid is to be understood herein as an organic acidhaving a pKa (logarithmic acid dissociation constant) of between −2 and12. Preferably the weak organic acid has a pKa of between 1 and 10, orbetween 2 and 5 or even between 3 and 4. The weak organic acid may be,but is not limited to, any carboxylic acid selected from the groupconsisting of oxalic acid (ethanedioic acid), citric acid(2-hydroxypropane-1,2,3-tricarboxylic acid), malic acid(2-hydroxybutanedioic acid), carbonic acid (hydroxymethanoic acid),benzoic acid (benzenecarboxylic acid or phenylmethanoic acid), formicacid (methanoic acid), lactic acid (2-hydroxypropanoic acid), aceticacid (ethanoic acid), butyric acid (butanoic acid), valeric acid(pentanoic acid), caproic acid (hexanoic acid), and propionic acid(propanoic acid). Most preferably, the organic acid is citric acid.

The organic acid may be present in the aqueous solution atconcentrations ranging from about 0.008 to 0.25M, or from about 0.01 to0.2M, or from 0.05 to 0.1M. The reconstitution composition of theinvention comprising 60-80%, or 65%-75% or 67%-72%, or about 70% of saidaqueous solution preferably has a resulting concentration of saidorganic acid ranging from 0.05 to 0.2M, 0.006 to 0.16M, 0.008 to 0.12M,0.03 to 0.08M, or preferably from 0.04 to 0.6M.

A lower alcohol is understood herein as an organic compound having ahydroxyl functional group bound to a saturated carbon atom of a loweralkyl or lower substituted alkyl group, wherein a lower alkyl or lowersubstituted alkyl group has at most 6 carbon atoms and preferably hasthe structure CH₃—(CH₂)_(n)—OH, wherein n=1, 2, 3, 4 or 5. Preferably,the lower alcohol is selected from the group consisting of methanol,ethanol, propanol, butanol and pentanol, most preferably the loweralcohol is ethanol.

The non-ionic hydrophilic surfactant preferably has ahydrophilic-lipophilic balance (HLB) value between 9 and 14, morepreferably between 12 and 14. This surfactant may be, but is not limitedto ethoxylated fatty acid mono- (in particular 5 ethoxyl groups), di- ortri- (in particular 20 ethoxyl groups) ester of sorbitan, wherein thefatty acid is preferably selected from the group consisting of oleate(e.g. ethoxylated sorbitan monooleate such as Tween 81® and/orethoxylated sorbitan trioleate such as Tween 85®), palmitate, stearate(e.g. ethoxylated sorbitan tristearate such as Tween 65®), isostearate,laurate and the combinations thereof; ethoxylated fatty alcohols (inparticular 5-10 ethoxyl groups) (e.g. Brij 76®, Brij 56®, Brij 96®),ethoxylated fatty acids (in particular 5-10 ethoxyl groups) (e.g.Simulsol 2599®, Myrj 45®), ethoxylated castor oil (in particular 25-35ethoxyl groups) (e.g. Arlatone 650®, Arlatone G®, Cremophor EL®), andcombinations thereof.

In one embodiment of the composition of the invention, the non-ionichydrophilic surfactant:

a. is a mono-, di or triglyceride, preferably an ethoxylatedtriglyceride, and/or

b. has a hydrophilic-lipophilic balance (HLB) value between 9 and 14.

The HLB value is calculated using the formula HLB=20 (1−I_(s)/I_(a)), inwhich L represents the saponification index or saponification value andI_(a) represents the acid index or acid value of said surfactant or ofsaid mixture of surfactants. These two indices, saponification and acidvalues, are determined by methods described in the EuropeanPharmacopoeia (Edition 8.8, section 2.5.6 and 2.5.1, respectively).

In a preferred embodiment, the non-ionic hydrophilic surfactants isethoxylated castor oil, more in particular polyoxyl 35 hydrogenatedcastor oil or polyoxyethyleneglyceroltriricinoleate 35 (CAS no.61791-12-6) (e.g. Cremophor EL®) which is a mixture of polyoxyethylatedtriglycerides obtained by reacting castor oil with ethylene oxide in amolar ration of 1:35.

Preferably, the reconstitution composition of the invention comprises orconsists of about 75% v/v aqueous solution comprising about 0.1M citricacid in water, about 6.25% v/v propylene glycol (CAS no. 57-55-6), about12.5% v/v ethanol and about 6.25% v/vpolyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6). In otherwords, the reconstitution composition comprises or consist of about0.075M citric acid, about 6.25% v/v propylene glycol (CAS no. 57-55-6),about 12.5% v/v ethanol and about 6.25% v/vpolyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6) in water.

Also preferred is a reconstitution composition comprising or consistingof about 75% v/v aqueous solution comprising about 0.1M citric acid inwater, about 6.25% v/v propylene glycol (CAS no. 57-55-6), about 12.5%v/v ethanol, about 6.25% v/v polyoxyethyleneglyceroltriricinoleate 35(CAS no. 61791-12-6) and 20 g/mL CpG ODN1826, or comprising orconsisting of about 0.075M citric acid, about 6.25% v/v propylene glycol(CAS no. 57-55-6), about 12.5% v/v ethanol, about 6.25% v/vpolyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6) and 20g/mL CpG ODN1826 in water.

The reconstitution composition is in particular suitable forreconstituting stored peptides as defined herein below underPharmaceutical composition, i.e. preferably having a length of between15 and 100 amino acids. The difficulty to form stable solutions uponreconstitution of peptides of the length defined above is appreciated inthe art, especially in case of different peptides, i.e. peptides havingdifferent amino acid sequences and hence have different chemicalproperties and behave physically different. As a result, it is hard toreconstitute them in one and the same solution. On top of that, in caseone or more of these peptides comprise cysteines, the tendency to formSS-bridges has to be dealt with. Although intramolecular disulfide bondsmay be required in vaccine peptides in order to be immunogenic,intermolecular disulfide bridge formation is undesirable as it resultsin instable solutions.

The inventors now have identified that the reconstitution composition ofthe present invention is in particular suitable for forming highlystable reconstituted peptides compositions wherein the amount ofintermolecular disulfide bridges is minimalized, without compromising onimmunogenicity of the reconstituted peptides. Therefore, thereconstitution composition of the invention prevents intermoleculardisulfide formation of the peptides to be reconstituted as furtherdefined herein, without comprising on immunogenicity of these peptides.

Preferably, the reconstitution composition of the invention is a sterileand/or pharmaceutical-grade or clinical-grade composition, suitable forparental administration to a subject, i.e. a mammalian species or humanbeing. Preferably, the reconstitution composition of the invention ismanufactured using Good Manufacturing Practice (GMP) and has GMP qualityas defined by both the European Medicines Agency and the Food and DrugAdministration. The reconstitution composition of the invention may bepackaged in a vial. The invention also provides for a vial comprising avolume of reconstitution composition suitable for reconstituting asingle pharmaceutical dosage unit as further defined herein, ormultiples thereof, i.e. a volume suitable for reconstituting a 2, 3, 4,5, 6, 7, 8, 9, 10 or more pharmaceutical dosage units. Preferably, saidvial is stored at a temperature at which the reconstitution compositionis stable for at least 1 month, 2 months, 3 months, 6 months or 1 yearor even 2 years. Preferably, said temperature is between −25° C. and 25°C., or between −23° C. and −18° C., or between 0° C. and 10° C., orbetween 2° C. and 8° C., or between 18° C. and 23° C.

Preferably, the volume of reconstitution composition of the inventionpresent in the vial is at most 50 mL, preferably between 0.1 and 10 mL,preferably between 1 and 10 mL, such as, 0.5, 1, 2, 3, 4, 5 or 10 mL, orany value in between. A vial is to be understood herein as a containerthat can have any shape. Optionally, a vial is to be understood hereinas a syringe.

Pharmaceutical Composition

The reconstitution composition of the invention is in particularsuitable for reconstituting peptides for the preparation of a medicamentor pharmaceutical composition. Such pharmaceutical composition may be avaccine, preferably a peptide vaccine. A “vaccine” is to be understoodherein as a composition comprising antigenic compounds, optionallycomplemented with further immune stimulating compounds, for generatingimmunity for the prophylaxis and/or treatment of diseases such asconditions associated with persistent infection and/or metaplasia and/ordysplasia and/or neoplasia. A “peptide-based vaccine” or “peptidevaccine” (these terms are used herein interchangeably) is to beunderstood herein as a vaccine wherein peptides constitute the activeingredients, i.e. the antigenic compounds. Preferably, such peptides aresynthetic long peptides. More preferably, comprising Human LeukocyteAntigen (HLA)-epitopes capable of inducing CD4+ and/or CD8+ T cellresponses.

Therefore, provided is a pharmaceutical composition comprising peptidesreconstituted in the reconstitution composition of the invention.Preferably, the pharmaceutical composition of the invention is avaccine, preferably a peptide-based vaccine. Such a peptide-basedvaccine may be used for the treatment of persistent infections,pre-cancerous conditions and cancer, preferably activating the cellularimmune system to clear infected, pre-cancerous and/or cancerous cellsthat express viral antigens, Tumor-Associated-Antigens, like cancertestis antigens and/or Tumor-Specific antigens, like oncogenic ornon-oncogenic viral antigens and/or neo-antigens resulting from DNAmutations.

The pharmaceutical composition is preferably for, and thereforeformulated to be suitable for, administration to a subject, preferably ahuman or animal subject. Preferably, the administration is parenteral,e.g. intravenous, subcutaneous, intramuscular, intradermalintracutaneous and/or intratumoral administration, i.e. by injection.

The inventors found that the reconstitution composition comprisingreconstituted peptides is in particular suitable for admixing with anoil-based adjuvant, resulting in a chemically and physically stablepeptide-vaccine solution.

“Chemically stable” is referred herein in the context of a peptidesolution and/or peptide-vaccine composition and is to be understoodherein as a solution or composition comprising peptides that do notchemically degrade or decompose, for instance because of the formationof intra- or intermolecular disulfide bridges, to an unacceptabledegree; i.e. the amount of un-degraded, un-decomposed and/or unreactedpeptides within the solution and/or composition is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% by weight ascompared to its original, after storage of the solution or compositionfor at least about 0.5, 1, 1.5, 2 or at least 3 hours at roomtemperature. Chemical stability can be assessed using any suitabletechnique known in the art, for instance using UPLC/MS as exemplifiedherein. When using UPLC/MS, a solution/composition is defined aschemically stable if the total % area of new peaks appearing afterstorage of at least about 0.5, 1, 1.5, 2 or at least 3 hours at roomtemperature is at most 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, or 0% as compared to its original, wherein new peaks are understoodto be the peaks on a UPLC chromatograms of the stored solution that werenot identified on the UPLC chromatograms of the original (“original”being understood herein as the freshly prepared solution directly afterpreparation), when measured under the same conditions. Preferably, thetotal % area of new peaks appearing after storage of 3 hours at roomtemperature is at most 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, or 0%, preferably at most 10% as compared to its original, whenmeasured under the same conditions.

“Physically stable” is referred herein in the context of a peptidesolution and/or peptide-vaccine composition and is to be understoodherein as a solution or composition comprising peptides that do notprecipitate or re-disperse. Physical stability can be assessed using anysuitable technique known in the art, for instance by visual inspectionor by particle distribution using a Malvern Mastersizer as exemplifiedherein, wherein average particle size is expressed in D(0.5). When usingMalvern Mastersizer for assessing physical stability as exemplifiedherein, a solution/composition is defined as physically stable if theaverage D (0.5) after storage of at least about 0.5, 1, 1.5, 2 or atleast 3 hours at room temperature is increased at most 50%, 40%, 30%,20%, 10% or 5% as compared to its original (i.e. the freshly preparedsolution directly after preparation). Preferably, a solution/compositionis defined as physically stable if the average D(0.5) after storage of 3hours at room temperature is increased at most 50%, 40%, 30%, 20%, 10%or 5%, preferably at most 20%, as compared to its original.

Preferably, the pharmaceutical composition of the invention furthercomprises an adjuvant. The term “adjuvant” is used herein to refer tosubstances that have immune-potentiating effects and are added to orco-formulated with an antigenic agent in order to enhance, induce,elicit, and/or modulate the immunological response against the antigenicagent when administered to a subject. Oil-based adjuvants can be used toform emulsions (e.g. water-in-oil or oil-in-water emulsions) and areappreciated in the art to enhance and direct the immune response. Thepresence of such adjuvant in a therapeutic vaccine is highly beneficial.Therefore, the present invention also provides for a pharmaceuticalcomposition or medicament comprising or consisting of the reconstitutioncomposition of the invention, reconstituted peptides and an oil-basedadjuvant, more in particular the invention provides for a pharmaceuticalcomposition comprising about 0.5-10 mg/mL peptides in about 40-60% v/vof the reconstitution composition of the invention and about 40-60% v/vof an oil-based adjuvant.

The oil-based adjuvant may be any mineral or non-mineral oil-basedadjuvant known in the art. Preferably the oil-based adjuvant is amineral oil-based adjuvant. Non-limiting examples of oil-based adjuvantsare bio-based oil adjuvants (based on vegetable oil/fish oil, etc.),squalene-based adjuvant (e.g. MF59), Syntex Adjuvant Formulation (SAF;Lidgate. Deborah A, Preparation of the Syntex Adjuvant Formulation (SSF,SAF-m, and SAF-1), In: Vaccine Adjuvants, Volume 42 of the seriesMethods in Molecular Medicine™ p 229-237, ISSN1543-1894), Freund'sComplete Adjuvant (FCA), Freund's Incomplete Adjuvant (FIA), adjuvantsbased on peanut oil (e.g. Adjuvant 65), Lipovant (Byars, N. E., Allison,A. C., 1990. Immunologic adjuvants: general properties, advantages, andlimitations. In: Zola, H. (Ed.), Laboratory Methods in Immunology. p39-51), ASO4 (A. Tagliabue, R. Rappuoli Vaccine adjuvants: the dreambecomes real Hum. Vaccine, 4 (5), 2008, p 347-349), Montanide adjuvants,which are based on purified squalene and squalene emulsified with highlypurified mannide mono-oleate (e.g. Montanide ISA 25 VG, 28 VG, 35 VG, 50V, 50 V2, 51 VG, 61 VG, 70 VG, 70 M VG, 71 VG, 720 VG, 760 VG, 763 A VG,775 VG, 780 VG, 201 VG, 206 VG, 207 VG). Preferably the oil-basedadjuvant is a mineral oil-based adjuvant. More preferably, the oil-basedadjuvant is Montanide ISA 51VG (Seppic), which is a mixture of DrakeolVR and mannide monooleate.

Preferably, the pharmaceutical composition comprises or consists of anamount of peptides that constitutes a pharmaceutical dosage unit. Apharmaceutical dosage unit is defined herein as the amount of activeingredients (i.e. the total amounts of peptides in a peptide-basedvaccine) that is applied to a subject at a given time point. Apharmaceutical dosage unit may be applied to a subject in a singlevolume, i.e. a single shot, or may be applied in 2, 3, 4, 5 or moreseparate volumes or shots that are applied preferably at differentlocations of the body, for instance in the right and the left limb.Reasons for applying a single pharmaceutical dosage unit in separatevolumes may be multiples, such as avoid negative side effects, avoidingantigenic competition and/or composition analytics considerations. It isto be understood herein that the separate volumes of a pharmaceuticaldosage may differ in composition, i.e. may comprise different kinds orcomposition of active ingredients and/or adjuvants. It is to beunderstood that for all active ingredients (antigenic peptides) withinthe whole pharmaceutical dosage unit a single reconstitution compositionis used, as one of the benefits of the invention is that thereconstitution composition of the invention is suitable forreconstituting, and subsequent emulsification using an oil-basedadjuvant, of different peptide mixtures. A single reconstitutioncomposition, and preferably a single oil-based adjuvant, minimizes thechance of human failure in reconstitution and emulsification.

A single injection volume or shot (i.e. volume applied on one locationat a certain time point), comprising a total pharmaceutical dosage, orpart thereof in case multiple shots applied at substantially the sametime point, may between 100 μL and 2 mL, or between 100 μL and 1 mL. Thesingle injection volume may be 100 μL, 200 μL, 300 μL, 400 μL, 500 μL,600 μL, 700 μL, 800 μL, 900 μL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL,1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 3 mL or any value inbetween.

A pharmaceutical dosage unit may be an effective amount or part of aneffective amount. An “effective amount” is to be understood herein as anamount or dose of active ingredients required to prevent and/or reducethe symptoms of a disease (e.g., chronic infection, pre-cancerouscondition and/or cancer) relative to an untreated patient. The effectiveamount of active compound(s) used to practice the present invention forpreventive and/or therapeutic treatment of a disease or condition variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount. This effective amountmay also be the amount that is able to induce an effective cellular Tcell response in the subject to be treated, or more preferably aneffective systemic cellular T cell response.

Preferably, pharmaceutical dosage unit, or total amount of peptidesapplied to a subject at a given time point, either in a single or inmultiple injections at a certain time point, comprises an amount ofpeptides in the range from 0.1 μg to 20 mg, such as about 0.1 μg, 0.5μg, 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg,80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg,450 μg, 500 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 1 mg,1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg,6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 15 mg or about20 mg or any value in between. Preferred ranges of pharmaceutical dosageunits are from 0.1 μg to 20 mg, 1 μg to 10 mg, 10 μg to 5 mg, 0.5 mg to2 mg, 0.5 mg to 10 mg or 1 mg to 5 mg or 2 to 4 mg.

Preferably, the pharmaceutical composition comprises or consists ofabout 1-2 mg/mL peptides in 40-60% v/v of the reconstitution compositionas defined above and 40-60% v/v of an oil-based adjuvant. Thepharmaceutical composition may comprise or consist about 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 56%, 57%,58%, 59% or 60% v/v of an oil-based adjuvant. Preferably, thepharmaceutical composition comprises or consists of about 1-2 mg/mLpeptides in about 50% v/v of the reconstitution composition as definedabove and about 50% v/v of an oil-based adjuvant, preferably MontanideISA 51 VG (Seppic). In other words, preferably, the pharmaceuticalcomposition comprises or consists of about 1-2 mg/mL peptide, 0.038Mcitric acid, about 3.13% v/v propylene glycol (CAS no. 57-55-6), about6.25% v/v ethanol, about 3.13% v/v polyoxyethyleneglyceroltriricinoleate35 (CAS no. 61791-12-6) and about 50% of an oil-based adjuvant,preferably Montanide ISA 51 VG (Seppic), in water.

The pharmaceutical composition of the invention may comprise one or morefurther immune response stimulating compounds or adjuvants.Advantageously, the medicament according to the invention mayadditionally comprise one or more synthetic adjuvants. Such a furtherimmune response stimulating compound or adjuvant may be (i) admixed tothe pharmaceutical composition according to the invention afterreconstitution of the peptides and optional emulsification with anoil-based adjuvant as defined above, (ii) may be part of thereconstitution composition of the invention defined above, (iii) may bephysically linked to the peptide(s) to be reconstituted or (iv) may beadministered separately to the subject, mammal or human, to be treated.It is to be construed herein that when an immune response stimulatingcompound is admixed to the medicament according to the invention, it isdepicted as an adjuvant; when administered separately, it is depicted asan immuno-modulatory agent, or an immuno-modulator, which terms are usedherein interchangeably. Particularly preferred are adjuvants that areknown to act via the Toll-like receptors and/or via a RIG-I (Retinoicacid-Inducible Gene-1) protein and/or via an endothelin receptor. Immunemodifying compounds that are capable of activation of the innate immunesystem can be activated particularly well via Toll like receptors(TLRs), including TLRs 1-10. Compounds capable of activating TLRreceptors and modifications and derivatives thereof are well documentedin the art. TLR1 may be activated by bacterial lipoproteins andacetylated forms thereof, TLR2 may in addition be activated by Grampositive bacterial glycolipids, LPS, LPA, LTA, fimbriae, outer membraneproteins, heat shock proteins from bacteria or from the host, andMycobacterial lipoarabinomannans. TLR3 may be activated by dsRNA, inparticular of viral origin, or by the chemical compound poly(I:C). TLR4may be activated by Gram negative LPS, LTA, Heat shock proteins from thehost or from bacterial origin, viral coat or envelope proteins, taxol orderivatives thereof, hyaluronan containing oligosaccharides andfibronectins. TLR5 may be activated with bacterial flagellae orflagellin. TLR6 may be activated by mycobacterial lipoproteins and groupB Streptococcus heat labile soluble factor (GBS-F) or Staphylococcusmodulins. TLR7 may be activated by imidazoquinolines, such as imiquimod,resiquimod and derivatives imiquimod or resiquimod (e.g. 3M-052). TLR9may be activated by unmethylated CpG DNA or chromatin—IgG complexes. Inparticular TLR3, TLR7 and TLR9 play an important role in mediating aninnate immune response against viral infections, and compounds capableof activating these receptors are particularly preferred in a thecompositions or medicaments according to the invention. Particularlypreferred adjuvants comprise, but are not limited to, syntheticallyproduced compounds comprising dsRNA, poly(I:C), poly I:CLC, unmethylatedCpG DNA which trigger TLR3 and TLR9 receptors, IC31, a TLR 9 agonist,IMSAVAC, a TLR 4 agonist, Montanide ISA-51, Montanide ISA 720 (anadjuvant produced by Seppic, France). RIG-I protein is known to beactivated by ds-RNA just like TLR3 (Kato et al, (2005) Immunity, 1:19-28). A particularly preferred TLR ligand is a pam3cys and/orderivative thereof, preferably a pam3cys lipopeptide or variant orderivative thereof, preferably such as described in WO2013051936A1, morepreferably U-Pam12 or U-Pam14 or AMPLIVANT®. Further preferred adjuvantsare Cyclic dinucleotides (CDNs), Muramyl dipeptide (MDP) and poly-ICLC.In a preferred embodiment, the adjuvants of the invention arenon-naturally occurring adjuvants such as the pam3cys lipopeptidederivative as described in WO2013051936A1, Poly-ICLC, imidazoquinolinesuch as imiquimod, resiquimod or derivatives thereof, CpGoligodeoxynucleotides (CpG-ODNs) having a non-naturally occurringsequence, and peptide-based adjuvants, such as muramyl dipeptide (MDP)or tetanus toxoid peptide, comprising non-naturally occurring aminoacids. Further preferred are adjuvants selected from the groupconsisting of: 1018 ISS, aluminum salts, Amplivax, AS 15, BCG,CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, ImuFact EV1P321,IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryllipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V,OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGAmicroparticles, SRL172, Virosomes and other Virus-like particles,Pam3Cys-GDPKHPKSF, YF-17D, VEGF trap, R848, beta-glucan, Aquila's QS21stimulon, vadimezan, AsA404 (DMXAA), STING (stimulator of IFN genes)agonist (e.g. c-di-GMP VacciGrade™), PCI, NKT (natural killer T cell)agonist (e.g. alpha-galactosylceramide or alpha-GalCer, RNAdjuvant®(Curevac), retinoic acid inducible protein I ligands (e.g. 3pRNA or5′-triphosphate RNA).

As indicated above, an adjuvant may be physically linked to thepeptide(s) to be reconstituted. Physical linkage of adjuvants andcostimulatory compounds or functional groups to antigenic peptides asdefined herein below provides an enhanced immune response by improvedtargeting to antigen-presenting cells, in particular dendritic cells,that internalize, metabolize and display antigen and by simultaneouslystimulating such cells to up-regulate expression of a variety ofco-stimulatory molecules, thereby becoming efficient T cell responseinducing and enhancing cells. Another preferred immune modifyingcompound is an inhibitor of an endothelin receptor such as BQ-788(Buckanovich R J et al., (2008) Nature Medicine 14: 28; Ishikawa K,(1994) PNAS 91: 4892), and/or derivatives thereof. BQ-788 isN-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine.Another preferred immune response stimulating compound or adjuvant isInterferon alpha (IFNα), more preferably pegylated Interferon alpha.Furthermore, the use of antigen presenting cell (co)stimulatorymolecules, as set out in WO99/61065 and in WO03/084999, in combinationwith the peptides and compositions of the invention is preferred. Inparticular the use of 4-1BB and/or CD40 ligands, agonistic antibodies,OX40 ligands, CD27 ligands or functional fragments and derivativesthereof, as well as synthetic compounds with similar agonistic activityare preferably administered separately or combined with the peptides ofthe invention to subjects to be treated in order to further stimulatethe mounting of an optimal immune response in the subject.

The peptides to be reconstituted in the reconstitution composition ofthe invention and/or comprised within the pharmaceutical composition ofthe invention, preferably have a length from about 15 to about 100 aminoacids. Preferably, the peptides to be reconstituted are between 15-100amino acids in length, or 15-95 amino acids, or 15-90 amino acids, or15-85 amino acids, or 15-70 amino acids, or 15-65 amino acids, or 15-60amino acids, or 15-55 amino acids, or 15-50 amino acids, or 15-45 aminoacids, or 15-40 amino acids, or 17-39 amino acids, or 19-43 amino acids,or 22-40 amino acids, or 22-45 amino acids, or 28-40 amino acids or30-39 amino acids in length. Preferably, the peptides to bereconstituted are at most 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90,89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72,71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54,53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,35, 34, 33, 32, 31, or 30 amino acids. Preferably, the peptides to bereconstituted are at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44 or 45 amino acids. Preferably, the peptides to be reconstituted areat least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acidsand no more than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88,87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70,69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52,51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34,33, 32, 31, or 30 amino acids, or any combination of these lower andupper limits.

The peptides to be reconstituted in the reconstitution composition ofthe invention and/or comprised within the pharmaceutical composition ofthe invention, may be peptides derived from protein antigens. A “proteinantigen” is to be understood herein as a protein or polypeptide thatcomprises antigenic regions capable of inducing an immune response in ahost animal or human. Protein antigens that are specifically expressedby infected, pre-cancerous and/or cancerous cells are suitable targetsfor therapeutic vaccines. Such protein antigens may be viral ornon-viral antigens. Examples of viral antigens that are targets forprophylactic and therapeutic vaccines are antigens derived from EpsteinBar virus induced lymphoma's (EBV), Human T lymphotrophic virus I,Hepatitis B virus (HBV), Human papilloma virus (HPV), Kaposi sarcomaherpes virus (KSHV), Hepatitis C virus (HVC), KSV and Merkel cellcarcinoma virus. Non-limiting examples of viral protein antigens areprotein antigens from EBV, e.g. LMP1 or late membrane protein 1 (e.g.UniprotKB P03230) and LMP2 or late membrane protein 2 (e.g. UniprotKBP13285); protein antigens from Human T lymphotrophic virus I, e.g. Taxprotein (e.g. UniprotKB P14079; POC213; P03409); protein antigens fromHBV e.g. genotypes A, B, C or D, e.g. protein HBsAg (e.g. UniprotKBQ773S4), X-protein (e.g. UniprotKB Q8V1H6) Large envelope protein (e.g.UniprotKB P03138) and capsid protein (e.g. UniprotKB P03147); proteinantigens from HCV, e.g. genome polyprotein (e.g. UniprotKB P26663;Q99IB8; A3EZI9) and HCV protein (e.g. UniprotKB Q99398); proteinantigens from HPV e.g. oncogenic genotypes 6, 11, 16 or 18, e.g., E6oncoprotein (e.g. UniprotKB P03126; P06463) and E7 oncoprotein (e.g.UniprotKB P03129; P06788) protein antigens from KSHV, e.g. protein ORF36(e.g. UniprotKB F5HGH5), Core gene UL42 family protein (e.g. UniprotKBQ77ZG5), Virion egress protein UL31 homolog (e.g. UniprotKB F5H982),Triplex capsid protein VP19C homolog (e.g. UniprotKB F5H8Y5), Viralmacrophage inflammatory protein 2 (e.g. UniprotKB Q98157), mRNA exportfactor ICP27 homolog (e.g. UniprotKB Q2HR75), ORF52 (e.g. UniprotKBF5HBL8), Viral IRF4-like protein (e.g. UniprotKB Q2HR73), Bcl-2 (e.g.UniprotKB Q76RI8), Large tegument protein deneddylase (e.g. UniprotKBQ2HR64), V-cyclin (e.g. UniprotKB 040946), VIRF-1 (e.g. UniprotKBF5HF68) and E3 ubiquitin-protein ligase MIR1 (e.g. UniprotKB P90495) andantigen protein Merkel cell carcinoma virus, e.g. large T protein (e.g.UniprotKB E2IPT4; K4P159), e.g. small T protein (e.g. UniprotKB B6DVX0;B6DVX6).

Non-viral antigens that are suitable targets for prophylactic andtherapeutic vaccines may be tumor specific antigens and/or tumorassociated antigen. Tumor specific antigens are antigens that areexclusively expressed by tumor cells and not by any other cell and areoften mutated proteins, such as KrasG^(12D) and mutant P53, orneo-antigens developed in due course by DNA mutations and malfunctioningDNA repair mechanisms. Tumor associated antigens are endogenous antigenspresent in both tumor and normal cells but are dysregulated in theirexpression or cellular localization, such as the HER-2/neu receptor. Nonlimiting examples of such non-viral antigens that may be targets fortherapeutic vaccines are Her-2/neu (or ErbB-2, Human Epidermal growthfactor Receptor 2 (e.g. UniprotKB P04626); WT-1 or Wilms tumor protein(e.g. UniprotKB P19544); NY-ESO-1 or cancer/testis antigen 1 (e.g.UniprotKB P78358); MAGE-A3 or melanoma-associated antigen-A3 (e.g.UniprotKB P43357); BAGE or B melanoma antigen (e.g UniProtKB Q13072);CEA or carcinoembryonic antigen (e.g UniProtKB Q13984); AFP orα-fetoprotein (e.g UniProtKB P02771); XAGE-1B or X antigen family member1 (e.g UniProtKB Q9HD64); survivin or BIRC5, Baculoviral IAPrepeat-containing protein 5 (e.g. UniprotKB 015392); P53 (e.g. UniprotKBP04637); h-TERT or Telomerase reverse transcriptase (e.g. UniprotKB014746); mesothelin (e.g. UniProtKB H3BR90); PRAME or Melanoma antigenpreferentially expressed in tumors (e.g. UniprotKB P78395); MUC-1 ormucin-1 (e.g. UniprotKB P15941); Mart-1/Melan-A or Melanoma antigenrecognized by T-cells 1 (e.g. UniprotKB Q16655); GP-100 or Melanocyteprotein PMEL (e.g. UniprotKB P40967); tyrosinase (e.g. UniprotKBU3M8N0); tyrosinase-related protein-1 (e.g. UniprotKB P17643);tyrosinase-related protein-2 (e.g. UniprotKB 075767); PAP or PAPOLA,Poly(A) polymerase alpha (e.g. UniprotKB P51003); PSA orProstate-specific antigen (e.g. UniprotKB P07288); PSMA orprostate-specific membrane antigen, or Glutamate carboxypeptidase 2(e.g. UniprotKB Q04609).

Preferred tumor specific antigen targets for peptide-vaccines are viraloncogenes and neo-antigens. “Neo-antigen” is to be understood herein asa tumor antigen that arises from a tumor-specific mutation(s) whichalters the amino acid sequence of genome-encoded proteins. Neo-antigenscan be identified by whole-genome sequencing elucidating all, or nearlyall, mutated neo-antigens that are uniquely present in a cancer (orneoplasia or tumor) of an individual patient. This collection of mutatedneo-antigens may be analyzed to identify a specific, optimized subset ofmutated neo-epitopes for use as an antigen source for the development ofa personalized cancer vaccine for treatment of the patient's cancer.Methods to identify such neo-antigens are described in WO2014/168874,which is incorporated herein by reference.

Peptides “derived” from an antigen protein is to be understood herein asto comprise a contiguous amino acid sequence selected from the antigenprotein, which may be modified by deletion or substitution of one ormore amino acids, by extension at the N- and/or C-terminus withadditional amino acids or functional groups, which may improvebio-availability, targeting to T-cells, or comprise or release immunemodulating substances that provide adjuvant or (co)stimulatoryfunctions.

The peptide to be reconstituted and/or comprised within thepharmaceutical composition may comprise or consist of a non-naturallyoccurring sequence as a result of the synthesis of non-natural lengthsor as a result of comprising additional amino acids not originating fromthe protein antigen where the peptide is derived for and/or as a resultof comprising a modified amino acid and/or a non-naturally occurringamino acid and/or a covalently linked functional group such as afluorinated group, a fluorocarbon group, a human toll-like receptorligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chainsor glycan, a pam3cys and/or derivative thereof preferably such asdescribed in WO2013051936A1, CpG oligodeoxynucleotides (CpG-ODNs),Cyclic dinucleotides (CDNs), a DC pulse cassette, a tetanus toxinderived peptide, a human HMGB1 derived peptide; either within thepeptide or appended to the peptide, as indicated above. The peptide ofthe invention may comprise 2-aminoisobutyric acid (Abu, an isostere ofcysteine). A cysteine of the peptide of the invention may be replaced byAbu.

Preferably, a peptide to be reconstituted and/or comprised within thepharmaceutical composition of the invention, is an isolated peptide,wherein “isolated” does not reflect the extent to which the peptide ispurified, but indicates that the peptide has been removed from itsnatural milieu (i.e., that has been subject to human manipulation), andmay be a recombinantly produced peptide or a synthetically producedpeptide.

The use of relatively short peptides is highly preferred for medicalpurposes as these can be efficiently synthesized in vitro, which is notpossible or uneconomical for native proteins larger than approximately100, i.e. 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 amino acids.Chemical synthesis of peptides is routine practice and various suitablemethods are known to the skilled person. Chemical synthesis of peptidesalso overcomes the problems associated with recombinant production ofintact proteins, which is difficult to standardize and requiresextensive purification and quality control measures. Peptides with alength that exceeds the length of human leukocyte antigen (HLA) class Iand class II epitopes (e.g. having a length as specified herein forpeptides to be reconstituted and/or comprised within the pharmaceuticalcomposition of the invention) are particularly advantageous for use asvaccine component because they are large enough to be taken up byprofessional antigen presenting cells (APC), in particular Dendriticcell (DC), as explained in WO02/070006, and processed in the DC beforecell surface presentation of the contained HLA class I-presented and HLAclass II-presented epitopes takes place. Therefore, the disadvantageousinduction of T cell tolerance by the systemic presentation of minimalHLA class I-presented epitopes on non-antigen presenting cells (as shownin Toes et al., Proc Natl Acad Sci (1996) USA 93(15):7855, and Toes etal., Immunol (1996) 156(10):3911), is prevented by the application ofpeptides exceeding the length of human leukocyte antigen (HLA) class Iand class II epitopes (as shown in Zwaveling et al., J. Immunol. (2002)169:350-358). As compared to vaccination with the peptides having alength as specified herein for peptides to be reconstituted and/orcomprised within the pharmaceutical composition of the invention,therapeutic vaccination with full length proteins are likely to be lesspotent (Rosalia et al. Eur. J Immunol (2013) 43: 2554-2565).

Peptides to be reconstituted and/or comprised in a pharmaceuticalcomposition of the invention are preferably peptides of about 15 toabout 100 amino acids in length, also denominated herein as longpeptides, that each exceed the length of human leukocyte antigen (HLA)class I and class II presented epitopes and that either on its own ormixed induce a combined CD4+ and CD8+ T cell response that istherapeutically successful and inducing cure in a high percentage ofpatients. Preferably, the long peptides of the invention are syntheticpeptides, denominated herein as synthetic long peptides (SLPs).

A “CTL epitope” is understood herein as a linear fragment of apolypeptide antigen that is liberated from the source protein byproteasome mediated proteolytic cleavage and subsequently presented byan HLA class I molecule on the cell surface of an antigen presentingcell (APC), preferably a human antigen presenting cell. A CTL epitope ofthe invention is preferably capable of activating a CD8⁺ T cellresponse. A CTL epitope typically comprises at least 8 up to 12, orexceptionally up to 13 or 14 amino acids. Preferably a CTL epitopeconsists of 8-14 amino acids, i.e. has a length of at least 8 up to 14amino acids.

A “Th-cell epitope” is understood herein to be a linear peptide fragmentthat is recognized by an HLA class II molecule. A Th-cell epitope iscapable of inducing a CD4⁺ T cell response. An HLA class II-restrictedCD4⁺ T-helper cell (Th-cell) epitope typically comprises 15 up to 20, orexceptionally even more, amino acids. Preferably, an HLA classII-restricted T-helper cell epitope comprises 10-20 or 10-15 aminoacids.

Most preferably, the Th-cell epitope of the peptide to be reconstitutedand/or comprises in the pharmaceutical composition of the invention, iscapable of activating a CD4⁺ T-helper memory and/or CD4⁺ T-helpereffector response, i.e. activation of a CD45RO-positive CD4⁺ T-helpercell. This will lead, by virtue of the ‘license to kill’ signal throughCD40-triggering of DC (Lanzavecchia (1998) Nature, 393: 413) to a morerobust CD8⁺ effector and memory cytotoxic T cell response. In anothersetting the activated CD4⁺ T-helper cells may activate non-HLArestricted killer cells of the immune system.

Within the context of the present invention “a peptide which comprisesat most 100 consecutive amino acids from a protein antigen” means thatthe number of consecutive amino acids originating from the proteinantigen and present in a peptide as defined herein, is 100, 99, 98, 97,96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79,78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61,60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43,42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids orless. Within the context of the present invention “a peptide whichcomprises at least 15 consecutive amino acids from a protein antigen”means that the number of consecutive amino acids originating from theprotein antigen and present in a peptide as defined herein, is at least15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more amino acids.Within the context of the present invention “a peptide which comprises15-100 consecutive amino acids from a protein antigen” means that thenumber of consecutive amino acids originating from the protein antigenand present in a peptide as defined herein, is at least 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids and no more than 100,99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82,81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64,63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46,45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 aminoacids. More preferably, the length of the contiguous amino acid sequencefrom the protein antigen comprised within the peptide to bereconstituted is 15-100 amino acids, or preferably 15-95 amino acids, or15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15-25amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 aminoacids, even more preferably 15-45 amino acids, even more preferably,15-40 amino acids, even more preferably 17-39, even more preferably19-43 amino acids, even more preferably 22-40 amino acids, even morepreferably 28-40 and even more preferably 30-39 amino acids.

Preferably, the pharmaceutical composition according to the inventiondoes not comprise any peptides which fulfill both of the followingproperties:

a. the percentage of basic amino acid residues equals the percentage ofacidic amino acid residues, and

b. the percentage of hydrophobic amino acid residues is 48% or higher.

For the purposes of this embodiment, amino acid residues are classifiedas “acidic”, “basic”, “hydrophobic” or “neutral” as follows:

Amino acid Category Asp Acidic Glu Acidic Arg Basic Lys Basic His BasicAla Hydrophobic Phe Hydrophobic Leu Hydrophobic Ile Hydrophobic ValHydrophobic Tyr Hydrophobic Trp Hydrophobic Cys Neutral Gly Neutral MetNeutral Pro Neutral Asn Neutral Gln Neutral Ser Neutral Thr Neutral

The peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention are preferably antigenicpeptides. “Antigenic peptides” are to be understood herein as (highly)immunogenic and capable of inducing a potent combined antigen-directedCD4+T helper and CD8+ cytotoxic T cell response, when administered as avaccine composition to a subject, preferably a human or animal subject.The peptide may be predicted to be immunogenic and/or may be proven tobe immunogenic using in vitro or ex vivo assays or by doing in vivotests appreciated in the art to establish immunogenicity. Preferably,the peptide can be used effectively in the prevention, partial clearanceand/or treatment or full clearance of an antigen related disease orcondition in a subject, preferably as detectable by:

-   -   activation or an induction of the immune system and/or an        increase in antigen specific activated CD4+ and/or CD8+ T-cells        in peripheral blood or in tissues as established by Elispot        assay or by tetramer staining of CD4+ or CD8+ T cells or an        increase of the cytokines produced by these T-cells as        established by intracellular cytokine staining of CD4+ and CD8+        T cells in flow cytometry after at least one week of treatment;        and/or    -   inhibition of proliferation of antigen related infection or a        detectable decrease of antigen expressing cells or a decrease in        cell viability of antigen expressing cells; and/or    -   induction or increased induction of cell death of antigen        expressing cells; and/or    -   inhibition or prevention of the increase of antigen expressing        cells.

In a preferred embodiment, a vaccine composition of the inventioncomprises a combination of peptides wherein said combination of peptidescovers at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of the HLA class I molecules that are encoded by HLA alleles predominantin the population of human subjects to be treated. HLA alleles that arepredominant in the population of human subjects to be treated. PreferredHLA class I epitopes in peptides according to the invention are epitopescapable of binding to: HLA-A0101; HLA-A0201; HLA-A0206; HLA-A0301;HLA-A1101; HLAA2301; HLA-A2402; HLA-A2501; HLA-A2601; HLA-A2902;HLA-A3001; HLAA3002; HLA-A3101; HLA-A3201; HLA-A3303; HLA-A6801;HLA-A6802; HLAA7401; HLA-B0702; HLA-B0801; HLA-B1301; HLA-B1302;HLA-B1402; HLAB1501; HLA-B1502; HLA-B1525; HLA-B1801; HLA-B2702;HLA-B2705; HLAB3501; HLA-B3503; HLA-B3701; HLA-B3801; HLA-B3901;HLA-B4001; HLAB4002; HLA-B4402; HLA-B4403; HLA-B4601; HLA-B4801;HLA-B4901; HLAB5001; HLA-B5101; HLA-B5201; HLA-B5301; HLA-B5501;HLA-B5601; HLAB5701; HLA-B5801 and HLA-B5802. In a preferred embodiment,a peptide of the invention, covers at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% of the HLA class I molecules that areencoded by HLA alleles predominant in the population of human subjectsto be treated, wherein “Cover an HLA class I molecule” is understoodherein as comprising a CTL epitope that shows binding affinity,preferably intermediate binding affinity, more preferably high bindingaffinity to said HLA class I molecule. Preferably, a peptide of theinvention covers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 100% of group of HLA class I molecules consisting of: HLA-A0101;HLA-A0201; HLA-A0206; HLA-A0301; HLA-A1101; HLA-A2301; HLA-A2402;HLA-A2501; HLA-A2601; HLA-A2902; HLA-A3001; HLA-A3002; HLA-A3101;HLA-A3201; HLA-A3303; HLA-A6801; HLA-A6802; HLA-A7401; HLA-B0702;HLA-B0801; HLA-B1301; HLA-B1302; HLA-B1402; HLA-B1501; HLA-B1502;HLA-B1525; HLA-B1801; HLA-B2702; HLA-B2705; HLA-B3501; HLA-B3503;HLA-B3701; HLA-B3801; HLA-B3901; HLA-B4001; HLA-B4002; HLA-B4402;HLA-B4403; HLA-B4601; HLA-B4801; HLA-B4901; HLA-B5001; HLA-B5101;HLA-B5201; HLA-B5301; HLA-B5501; HLA-B5601; HLA-B5701; HLA-B5801 andHLA-B5802.

The reconstitution composition can be used for reconstituting a singletype of peptides (i.e. all having substantially the same, or the sameamino acid sequence) or for mixtures of different peptides havingdifferent amino acid sequences. A pharmaceutical composition of theinvention preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32 and up to 33 different peptides. “Different peptides” are tobe understood herein as having a different amino acid sequences,preferably having less than 60%, 50%, 40%, or preferably less than 30%sequence identity, as determined over their whole length. The differentpeptides to be reconstituted and/or comprised within the pharmaceuticalcomposition of the invention may be peptides having a length as definedherein above that together overlap the entire amino acid sequence of theprotein antigen from which these peptides are derived. However, in someinstances, immunization with the complete set of overlapping (synthetic)long peptides spanning the full length protein antigens is not feasible,and a selection needs to be made. To narrow the number of peptides in avaccine, preferably the most immunogenic long peptides are selected andincorporated that are recognized by the highest percentage of patients.

At least one of the peptides to be reconstituted and/or comprised withinthe pharmaceutical composition of the invention may have at least onecysteine residue that is capable of intermolecular disulfide bridging,or may have at least two cysteine residues that are capable of intra-and inter-molecular disulfide bridge formation. Preferably, a vaccinecomposition according to the invention comprises a combination ofpeptides wherein said combination of peptides covers at least 70%, 80%,90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the HLA class Imolecules that are encoded by HLA alleles predominant in the populationof human subjects to be treated as defined herein above.

The amount of peptides to be reconstituted in the reconstitutioncomposition of the invention preferably is a pharmaceutical dosage unitand/or amount to be injected in a single volume, as defined hereinabove.

Dried peptides may be peptides free of further constituents but may alsocomprise buffer components such as Trifluor acetic acid (TFA), saltssuch as sodium, potassium or phosphate salts (e.g. NaCl, KCl and NaPO₄).The amount of further constituents is preferably less than 30%, morepreferably less than 25%, of the total weight of the dry peptides to bereconstituted. Dried peptides to be reconstituted may be in a physicaldried state as can be obtained by processes such as, but not limited to,rotor evaporation, lyophilization (freeze drying) and spray drying.

Preferred protein antigens, from which the peptides to be reconstitutedand/or comprised within the pharmaceutical composition of the inventionare derived, are defined herein below.

HPV-Derived Peptides

The peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention may be (mixes of) peptidesderived from the early HPV antigen proteins E2, E6 or E7. Preferably,the contiguous amino acid sequence is selected from the full lengthamino acid sequences of the HPV E6 and E7 proteins from a high risk HPVserotype, such as serotypes 16, 18, 31, 33 or 45, more preferably fromthe amino acid sequences of the HPV E6 and E7 serotypes 16, 18, 31 or33, most preferably from serotypes 16 or 18, of which 16 is mostpreferred. The amino acid sequence of the HPV serotype 16 E2(UniProtKB—P03120), E6 (UniProtKB—P03126) and E7 (UniProtKB—P03129)proteins are depicted in SEQ ID NO: 14-16, respectively. The amino acidsequence of the HPV serotype 18 E2 (UniProtKB—P06790), E6(UniProtKB—P06463) and E7 (UniProtKB—P06788) proteins are depicted inSEQ ID NO: 17-19, respectively.

Preferred peptides and peptide mixes to be reconstituted and/orcomprised within the pharmaceutical composition of the invention andderived from HPV E6 and E7 proteins are as defined in WO00/75336 A2.Preferred peptides are peptides comprising or consisting of a contiguoussequence within an immunogenic region represented by any one of SEQ IDNO: 20-26.

Preferably, one or more peptides to be reconstituted and/or comprisedwithin the pharmaceutical composition of the invention comprises a CTLepitope selected from the group represented by SEQ ID NO: 27-67.

Preferred peptides and peptide mixes to be reconstituted and/orcomprised within the pharmaceutical composition of the invention andderived from HPV E2, E6 and E7 proteins are as defined in WO2002/070006A2 and WO2002/090382, which is incorporated herein by reference.Preferred peptides are peptides comprising or consisting of a contiguoussequence within the following HPV immunogenic regions E2 (31-120); E2(151-195); E2 (271-365); E6 (81-158); E7 (31-77), preferably of theHPV16 serotype and defined herein by SEQ ID NO: 68-72, respectively.

Preferably, one or more peptides to be reconstituted and/or comprisedwithin the pharmaceutical composition of the invention comprises a Thepitope that is selected from DR1/E2 351-365, DR2/E2 316-330, DR2/E2346-355, DR4/E2 51-70, E2 61-76, DQ6/E2 311-325, DR15/E7 50-62, DR3/E743-77, DQ2/E7 35-50 and DR1/E6 127-142 (represented herein by SEQ ID NO:73-82, respectively).

Preferably, the peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention comprise at least one T cellepitope that is recognized by a T cell that infiltrates a cervicalneoplastic lesion or by a T cell that is present in or isolated from alymph node from the pelvic region, that is draining from the cervicalneoplastic lesion. Preferably, the T cell epitope is present in orisolated from a draining lymph node comprising metastatic tumor cells.Such epitopes are disclosed in e.g., WO2008/147187 A1, US20060182762A1,WO2006013336A1, WO2009148230A2, WO2009148229A2, WO2002044384A2 which isincorporated herein by reference.

In yet a preferred peptide to be reconstituted and/or comprised withinthe pharmaceutical composition of the invention, the contiguous aminoacid sequence comprises an epitope that is selected from the groupconsisting of a contiguous amino acid sequence selected from the groupconsisting of the amino acid sequences represented by SEQ ID NO: 83-104,which have been proven to be T-cell epitopes that are recognized by a Tcells that infiltrates a cervical neoplastic lesion or by a T cell froma draining lymph node.

A preferred class II CD4⁺ Th cell epitope comprised in a peptide to bereconstituted and/or comprised within the pharmaceutical composition ofthe invention is selected from the group consisting a contiguous aminoacid sequence selected from the group consisting of the amino acidsequences represented by SEQ ID NO: 83-99.

A preferred class I CD8⁺ CTL cell epitope comprised in a peptide to bereconstituted and/or comprised within the pharmaceutical composition ofthe invention is selected from the group consisting a contiguous aminoacid sequence selected from the group consisting of the amino acidsequences represented by SEQ ID NO: 85, 82, 100-104.

Preferred peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention comprise or consist of acontiguous amino acid sequence selected from the group consisting of theamino acid sequences represented by SEQ ID NO: 1-13.

Preferred mixes of peptides to be reconstituted and/or comprised withina vaccine composition of the invention are mixes of peptides that haveat least 1, 2, 3, 4 or 5 of the peptides comprising or consisting of thesequences selected from SEQ ID NO: 1-5; at least 1, 2, 3, 4, 5 or 6 ofthe peptides comprising or consisting of a contiguous amino acidsequence selected from the group consisting of the amino acid sequencesrepresented by SEQ ID NO: 1-6; and at least 1, 2, 3, 4, 5, 6 or 7 of thepeptides comprising or consisting of a contiguous amino acid sequenceselected from the group consisting of the amino acid sequencesrepresented by SEQ ID NO: 7-13. Preferably, the pharmaceuticalcomposition comprises a mixture of peptides having sequence SEQ ID NO:1-5 or SEQ ID NO: 1-6 or SEQ ID NO: 7-13. Preferably, the differentpeptides in the mixture are present in the pharmaceutical composition insubstantially equal ratios.

HBV-Derived Peptides

The peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention may be (mixes of) peptidesderived from the various genotypes, e.g. from the HBV-A proteins, HBVpolymerase (UniProtKB—P03159), HBV core protein (UniProtKB—POC625), HBVX protein, and HBV large surface protein (UniProtKB—P03141), which arerepresented herein by SEQ ID NO: 105-108. Preferred peptides, peptidesmixes and epitopes present within these peptides have been disclosed ine.g. WO2015/187009, WO2014/102540 A1, WO 93/03753, WO 95/03777,US2010/0068228A1, US2009/0311283 A1, which are incorporated herein byreference.

Preferred, one or more peptides to be reconstituted and/or comprisedwithin the pharmaceutical composition of the invention comprise orconsist of an amino acid sequence selected from the group consisting ofSEQ ID NO: 109-146.

Preferably, the peptide to be reconstituted and/or comprised within thepharmaceutical composition of the invention comprises or consist of apeptide selected from the group consisting of SEQ ID NO: 109, 113, 118,121, 122, 126, 129, 132, 133, 134, 135, 138 and 142, more preferablyselected from the group consisting of SEQ ID NO: 109, 113, 118, 121,122, 126, 129, 132, 133, 135, 138 and 142, even more preferably selectedfrom the group consisting of SEQ ID NO: 113, 118, 121, 122, 126, 129,132, 133, 134, 135 and 142, even more preferably selected from the groupconsisting of SEQ ID NO: 113, 118, 121, 122, 126, 129, 132, 133, 135 and142, even more preferably selected from the group consisting of SEQ IDNO: 118, 121, 129, 132, 133 and 142, most preferably selected from thegroup of SEQ ID NO: 133, 142 and 121. Preferably, the one or morepeptides to be reconstituted and/or comprised within the pharmaceuticalcomposition of the invention comprises or consists of a peptide selectedfrom the group consisting of SEQ ID NO: 109, 118, 121, 122, 126, 129,132-135. Preferably, the one or more peptides to be reconstituted and/orcomprised within the pharmaceutical composition of the inventioncomprises or consists of a peptide selected from the group consisting ofSEQ ID NO: 122, 129 and 133.

Preferred mixes of peptides to be reconstituted and/or comprised withina vaccine composition of the invention are mixes of peptides at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different peptidesof the peptides consisting of or comprising of a peptide selected fromthe group consisting of SEQ ID NO: 109-146, more preferably selectedfrom the group consisting of SEQ ID NO: 109, 113, 118, 121, 122, 126,129, 132, 133, 134, 135, 138 and 142, more preferably selected from thegroup consisting of SEQ ID NO: 109, 113, 118, 121, 122, 126, 129, 132,133, 135, 138 and 142, even more preferably selected from the groupconsisting of SEQ ID NO: 113, 118, 121, 122, 126, 129, 132, 133, 134,135 and 142, even more preferably selected from the group consisting ofSEQ ID NO: 113, 118, 121, 122, 126, 129, 132, 133, 135 and 142, evenmore preferably selected from the group consisting of SEQ ID NO: 118,121, 129, 132, 133 and 142, most preferably selected from the group ofSEQ ID NO: 133, 142 and 121. Further preferred is a composition thatcomprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to33 different peptides of the peptides consisting of or comprising of apeptide selected from the group consisting of SEQ ID NO: 109, 118, 121,122, 126, 129, 132-135, more preferably selected from the groupconsisting of SEQ ID NO: 121, 129 and 133. Further preferred mixes to bereconstituted and/or comprised within the pharmaceutical composition ofthe invention are a mix comprising a peptide that comprises or consistsof a peptide of SEQ ID NO: 121 in combination with a peptide thatcomprises or consists of at least one of SEQ ID NO: 139, 140, 133, 139,142, 118, 129; and a mix comprising a peptide that comprises or consistsof a peptide of SEQ ID NO: 133 in combination with a peptide thatcomprises or consists of at least one of SEQ ID NO: 139, 140, 63, 139,142, 118, 129. Preferably, the different peptides in the mixture arepresent in the pharmaceutical composition in substantially equal ratios.

PRAME-Derived Peptides

The peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention may be (mixes of) peptidesderived from PRAME (UniProtKB—P78395), which is represented herein bySEQ ID NO: 147. Preferred peptides, peptides mixes and epitopes presentwithin these peptides have been disclosed in e.g., WO 2008/118017 A2which is incorporated herein by reference. Preferably, one or more ofthe peptides to be reconstituted comprise or consist a peptide selectedfrom the group consisting of the amino acid sequence defined by SEQ IDNO: 148-167. Preferred mixes of peptides to be reconstituted and/orcomprised within a vaccine composition of the invention are mixes atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 different peptides selected from SEQ ID NO: 148-167.

Preferably, the one or more peptides to be reconstituted and/orcomprised within the pharmaceutical composition of the inventioncomprises a Th epitope that is selected from SEQ ID NO: 168-169.

P53-Derived Peptides

The peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention may be (mixes of) peptidesderived from P53 (e.g. UniprotKB P04637), which is represented herein bySEQ ID NO: 190. Preferred peptides, peptides mixes and epitopes presentwithin these peptides have been disclosed in e.g., WO 2008/147186 A2,which is incorporated herein by reference. Preferably, one or more ofthe peptides to be reconstituted comprise or consist a peptide selectedfrom the group consisting of the amino acid sequence defined by SEQ IDNO: 191-211, more preferably selected from the group consisting of theamino acid sequence defined by SEQ ID NO: 191-204.

Preferred mixes of peptides to be reconstituted and/or comprised withina vaccine composition of the invention are mixes at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different peptidesselected from SEQ ID NO: 191-211.

PSMA-Derived Peptides

The peptides to be reconstituted and/or comprised within thepharmaceutical composition of the invention may be (mixes of) peptidesderived from PSMA (e.g. UniprotKB Q04609), which is represented hereinby SEQ ID NO: 212. Preferred peptides, peptides mixes and epitopespresent within these peptides have been disclosed in e.g., WO2013/006050 A1, which is incorporated herein by reference. Preferably,one or more of the peptides to be reconstituted comprise or consist apeptide selected from the group consisting of the amino acid sequencedefined by SEQ ID NO: 213-232.

Preferred mixes of peptides to be reconstituted and/or comprised withina vaccine composition of the invention are mixes at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different peptidesselected from SEQ ID NO: 213-232.

Also comprised within the preferred antigen proteins, peptides to bereconstituted and epitopes within these peptides are antigen proteins,peptides and epitopes that show substantial identity to any of thespecific antigen proteins, peptides and epitopes defined herein.Sequence identity is herein defined as a relationship between two ormore amino acid sequences (polypeptide or protein), as determined bycomparing the sequences. In the art, “identity” also means the degree ofsequence relatedness between amino acid sequences as determined by thematch between strings of such sequences. Sequence identity can bedetermined by alignment of two peptide sequences using global or localalignment algorithms, depending on the length of the two sequences.Sequences of similar lengths are preferably aligned using a globalalignment algorithms (e.g. Needleman Wunsch) which aligns the sequencesoptimally over the entire length, while sequences of substantiallydifferent lengths are preferably aligned using a local alignmentalgorithm (e.g. Smith Waterman). Sequences may then be referred to as“substantially identical” when they (when optimally aligned by forexample the programs GAP or BESTFIT using default parameters) share atleast a certain minimal percentage of sequence identity (as definedbelow). GAP uses the Needleman and Wunsch global alignment algorithm toalign two sequences over their entire length (full length), maximizingthe number of matches and minimizing the number of gaps. A globalalignment is suitably used to determine sequence identity when the twosequences have similar lengths. Generally, the GAP default parametersare used, with a gap creation penalty=50 (nucleotides)/8 (proteins) andgap extension penalty=3 (nucleotides)/2 (proteins). For proteins thedefault scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89,915-919). Sequence alignments and scores for percentage sequenceidentity may be determined using computer programs, such as the GCGWisconsin Package, Version 10.3, available from Accelrys Inc., 9685Scranton Road, San Diego, Calif. 92121-3752 USA, or using open sourcesoftware, such as the program “needle” (using the global NeedlemanWunsch algorithm) or “water” (using the local Smith Waterman algorithm)in EmbossWIN version 2.10.0, using the same parameters as for GAP above,or using the default settings (both for ‘needle’ and for ‘water’, thedefault Gap opening penalty is 10.0 and the default gap extensionpenalty is 0.5; default scoring matrices are Blossum62 for proteins).When sequences have a substantially different overall length, localalignments, such as those using the Smith Waterman algorithm, arepreferred.

Alternatively, percentage identity may be determined by searchingagainst public databases, using algorithms such as FASTA, BLAST, etc.Thus, the protein sequences of the present invention can further be usedas a “query sequence” to perform a search against public databases to,for example, identify other family members or related sequences. Suchsearches can be performed using the BLASTp and BLASTx programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST proteinsearches can be performed with the BLASTx program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTx and BLASTp) can be used. See the homepage of the NationalCenter for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

An antigen protein, peptide or epitope that show substantial identity toits related antigen protein, peptide or epitope defined herein is to beunderstood herein to have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the specificsequences referred to herein, based on the full length of said specificsequence (i.e. over its whole length or as a whole).

Medical Use

Provided is a method for preventing or treating a persistent or chronicinfection, pre-cancerous disorder and/or cancer. In other words,provided is the pharmaceutical composition of the invention as definedherein above for use as a medicament, preferably for the prevention ortreatment of a persistent or chronic infection, pre-cancerous disorderand/or cancer. Such method or use comprises the step of administratingthe pharmaceutical composition of the invention to a subject that is inneed of such prevention and/or treatment. A subject in need ofprevention and/or treatment may also be referred to as a patient, andmay refer to an animal such as a mammal, including, but not limited to,a human or a non-human mammal, such as a non-human primate, bovine,equine, canine, ovine, or feline.

Preferably, a pharmaceutical dosage unit as defined herein above isprovided. As also indicated herein above, this pharmaceutical dosageunit may be given once in a single shot or as multiple volumesadministered at different locations. For example, a pharmaceuticaldosage unit may be divided over two shots each administered in one ofthe two legs or arms of the subject to be treated. The two shots maycomprise the same or different peptides mixes. For instance, a firstshot may comprise SEQ ID NO 1-5 or SEQ ID NO: 1-6 and a second shot maycomprise SEQ ID NOs 7-13, wherein both shots are administered at asingle or substantially single time point, wherein substantially singletime point is to be understood as within at most about 15 minutes,preferably, within at most 2 minutes.

The administration of the single or multiple shot may be carried outonce or alternatively may be repeated subsequently, such as, but notlimited to, daily, bi-weekly, weekly, once every two weeks, once everythree weeks, once every four weeks, once every five weeks, once everysix weeks, monthly, once per 2 months, once per 3 months, once per 4months, once per 5 months, once per 6 months, once per 7 months, onceper 8 months, once a year, once per 2 years, once per 5 years or onceper 10 years.

Preferably, the pharmaceutical composition is administered in aneffective amount as defined herein above. Preferably, the pharmaceuticalcomposition of the invention is for intravenous or subcutaneous, orintramuscular administration, although other administration routes canbe envisaged, such as mucosal administration or intradermal and/orintracutaneous administration or intratumoral administration, e.g., byinjection. The pharmaceutical composition of the invention may beadministered by a single administration. Alternatively, theadministration may be repeated if needed and/or distinct peptides orpeptide mixes or composition comprising different peptides or peptidemixes may be sequentially administered, wherein sequentially may be intime and/or location.

Preferably, the pharmaceutical composition is a vaccine composition forinducing a T cell response against at least one epitope comprised in apeptide. Preferably, the vaccine is for the prevention, partialclearance and/or treatment or full clearance of a antigen associateddisease or condition in a subject, e.g. a persistent infection,cancerous (neoplasia) or precancerous disorder, preferably as detectableby:

-   -   activation or an induction of the immune system and/or an        increase in antigen specific activated CD4+ and/or CD8+ T-cells        in peripheral blood or in tissues as established by Elispot        assay or by tetramer staining of CD4+ or CD8+ T cells or an        increase of the cytokines produced by these T-cells as        established by intracellular cytokine staining of CD4+ and CD8+        T cells in flow cytometry after at least one week of treatment;        and/or    -   inhibition of proliferation of antigen related infection or a        detectable decrease of antigen expressing cells or a decrease in        cell viability of antigen expressing cells; and/or    -   induction or increased induction of cell death of antigen        expressing cells; and/or    -   inhibition or prevention of the increase of antigen expressing        cells.

Examples of cancers to be prevented and/or treated via a method of theinvention include, without limitation, cervical intraepithelialneoplasia (CIN), Vulvar intraepithelial neoplasia (VIN), vaginalintraepithelial neoplasia (VaIN), anal intraepithelial neoplasia (AIN),and penal intraepithelial neoplasia (PIN), as well as cancer of thecervix, vulva, vagina, anus, penis, aerodigestive track, and head &neck; liver cancer, leukemia (e.g., acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma(e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors such assarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, uterine cancer, testicularcancer, lung carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, bladder carcinoma, multiple myeloma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

The method of the invention may be part of a combination therapy, whichmay be provided as a separate treatment or added to the pharmaceuticalcomposition of the invention. The method of the invention may becombined with checkpoint control blockers, monoclonal antibodies (mAbs)targeting selected TNF receptor family members (e.g. CD40, 4-1 BB/CD137,OX-40/CD134, and CD27), immunosuppressive cytokines (e.g. IL-10, TGF-βand IL-6) and/or yC cytokines (e.g. IL-7, IL-15, and IL-21 or IL-2), IDO(indoleamine 2,3-dioxygenase) inhibitors, thalidomide and/or derivativesthereof, further immunomodulators (e.g. compounds that are known todeplete immunosuppressive Tregs and/or MDSCs), standard of caretreatment, e.g. chemotherapy, radiotherapy, surgery, IFN-α conditioning,antiviral therapy, antibacterial therapy, UV therapy, anti-inflammatorytherapy, etc. In case of the treatment or prevention of a pre-cancerousdisorder or a cancer, the peptide-based vaccine may be combined withradiotherapy and/or chemotherapy such as treatment with carboplatin,paclitaxel, CarboTaxol (a combination of carboplatin, paclitaxel) and/orcisplatin. For example, the method of the invention may be part of achemotherapy regimen wherein chemotherapy is applied once every threeweeks. Preferably, a first pharmaceutical dosage unit of apharmaceutical composition of the invention is administered 2 weeksafter the second or third cycle of chemotherapy.

Method for Reconstitution

Also provided is a method for reconstituting dried, preferablylyophilized, peptides, comprising the following subsequent steps:

-   -   a) providing a vial comprising dried, preferably lyophilized,        peptides;    -   b) thawing the peptides, preferably for about 5-30 min;    -   c) adding the reconstitution composition of the invention to the        vial comprising the peptides, preferably without swirling the        vial;    -   d) allowing to admix, preferably for about 0.5-5 minutes; and    -   e) swirling until a clear solution is obtained, preferably for        about 1-3 minutes.

Preferably, steps b) to e) are performed at room temperature.

Further provided is a method for preparing a pharmaceutical composition,comprising the subsequent steps of:

-   -   (i) collect reconstituted peptides obtainable by the method for        reconstituting dried peptides as defined above in a first        syringe;    -   (ii) connect the first syringe of step (i) to a second syringe        comprising the oil-based adjuvant using a connector;    -   (iii) push the content of the first syringe into the second        syringe and backwards    -   (iv) repeat step (iii) about 10-50 times in a total in about        10-50 seconds.

Preferably, steps (i) to (iv) are performed at room temperature.

The clear solution obtained in step e) in the method for reconstitutingdried peptides is to be understood herein as a reconstitutioncomposition comprising reconstituted peptides, which can be used asstarting material, i.e. as “reconstituted peptides” in step (i) of amethod for preparing a pharmaceutical composition.

Preferably, the dried, preferably lyophilized, peptides in the vial andused as starting material in step a) in the method for reconstitutingdried peptides, are peptides as defined herein above as peptides to bereconstituted and/or peptide to be comprised in the pharmaceuticalcomposition of the invention. Preferably, said vial comprises peptidesin an amount for injection as a single volume in a method for preventionand/or treatment, preferably a method of treatment and/or prevention asdefined herein, i.e. a single pharmaceutical dosage unit, or partthereof in case of multiple injections at difference locations of thesubjects body at substantially the same time point. Alternatively, theamount of dried peptides in the vial in step a) is exceeding the amountfor injection as a single volume in said method. For instance, theamount of peptides within the vial may be twice the amount for injectionas a single volume. In the latter case, half of the amount ofreconstituted volume may be admixed with an amount of oil-based adjuvantin a method for preparing a pharmaceutical composition such as thepharmaceutical composition of the invention, in order to end up with asingle volume of pharmaceutical composition for injection in a method ortreatment or prevention, or, alternatively, the total amount ofreconstituted volume may be admixed with an amount of oil-based adjuvantin order to end up with two volumes of pharmaceutical composition forinjection.

Preferably, the peptides in step b) of the method to reconstitutepeptides, are thawed at room temperature for about 5-30 min, or 10-30min, such as for 5, 10, 15, 20, 25 or 30 minutes, or any value inbetween.

Preferably, the admixing in step d) of the method to reconstitutepeptides, is without substantially swirling or stirring the vialpreferably for about 0.5-2 minutes at room temperature, such as for 0.5,1, 1.5 or 2 minutes. In other words, preferably the peptides in step d)is allowed to admix with the reconstitution composition while standingstill.

The swirling in step e) of the method to reconstitute peptides, isperformed by swirling until a clear solution is obtained. As indicated,this is performed preferably for about 1-3 minutes. However, for somepeptides longer or shorter swirling time is required. However, a clearsolution should preferably be obtainable within 20 minutes. Therefore,the swirling may be performed in a range from 1 to 20 min, from 1 to 10min, from 1 to 5 minutes or from 1 to 3 minutes, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 minutes, or any value in between, preferablyuntil a clear solution is obtained upon visual inspection.

The reconstitution composition of step c) of the method forreconstituting peptides, and the oil-based adjuvant of step (ii) of themethod for preparing a pharmaceutical composition, are as definedearlier herein. Preferably, the amount of reconstitution composition instep c) is in a range of from about 0.5 and 2 mL, preferably 1 mL.Preferably, the amount of reconstituted peptides in step (i) is thetotal amount of reconstituted peptides as obtained after step e), i.e.within the clear solution obtained after step e). However, optionallyless is used, as exemplified above. Preferably, the volume of thisreconstitution composition is admixed with oil-based adjuvant in step(ii) to (iv) in a ratio of about 2:1 to about 1:2, such as 2:1, 1.9:1,1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.1:1, 1:1, 1:1.9, 1:1.8, 1:1.7,1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, or 1:1.1, preferably 1:1 ofreconstitution composition:oil-based adjuvant.

The connector in (ii) may be any connector suitable in the art toconnect two syringes that allow fluids to be exchanged between the twosyringes, such as but not limited to, T and I connectors. The repeats(iv) may be about 10-50 times, such as, but are not limited to, 10, 15,20, 25, 30, 45, 50 times, or any value in between.

Kit of Parts

Further, provided is a kit of parts comprising a:

-   1. a first vial containing dried, preferably lyophilized, peptides,    wherein preferably the peptides are peptides as defined herein    above;-   2. a second vial containing a reconstitution composition of the    invention; and, optionally,-   3. a third vial containing an oil-based adjuvant, preferably as    defined herein above.

Preferably, the all components, i.e. dried peptides, reconstitutioncomposition and oil-based adjuvant, are sterile and/orpharmaceutical-grade or clinical-grade. Preferably, these components aremanufactured using Good manufacturing practice (GMP) and have GMPquality as defined by both the European Medicines Agency and the Foodand Drug Administration.

Preferably, the first vial is stored at a temperature at which thereconstitution composition is stable for at least 1 month, 2 months, 3months, 6 months or 1 year or even 2 years. Preferably, said temperatureis between −25° C. and 25° C., or between −23° C. and −18° C., orbetween 0° C. and 10° C., or between 2° C. and 8° C., or between 18° C.and 23° C. Preferably, the second vial is stored at a temperature atwhich the reconstitution composition is stable for at least 1 month, 2months, 3 months, 6 months or 1 year or even 2 years. Preferably, saidtemperature is between −25° C. and 25° C., or between −23° C. and −18°C., or between 0° C. and 10° C., or between 2° C. and 8° C., or between18° C. and 23° C. . . . Preferably, the third vial is stored at atemperature at which the reconstitution composition is stable for atleast 1 month, 2 months, 3 months, 6 months or 1 year or even 2 years.Preferably, said temperature is between −25° C. and 25° C., or between−23° C. and −18° C., or between 0° C. and 10° C., or between 2° C. and8° C., or between 18° C. and 23° C. Preferably, the first, second andthird vial are stored at the same temperature.

Optionally, said kit of parts further comprises a manual describing themethod for reconstituting dried peptides as defined herein above,storage conditions, a method for preparing a pharmaceutical compositionas defined herein above and/or a manual for storing the first, secondand/or third vial. In addition, the kit of parts may comprise a manualfor administering the pharmaceutical composition to be prepared.Preferably, the volume of the first, second and/or third vial is at most50 mL, preferably between 0.1 and 10 mL, preferably between 1 and 10 mL,such as, 0.5, 1, 2, 3, 4, 5 or 10 mL, or any value in between. A vial isto be understood herein as a container that can have any shape.Optionally a vial is to be understood herein as a syringe. Optionally,the first vial can be connected via a connector by an active handlingprocess to the second vial to allow the reconstitution composition tocontact and dissolute the peptides. Optionally, the second vial cansubsequently be connected to the third vial to allow the reconstitutioncomposition comprising the peptides to be admixed with the oil-basedadjuvant. Optionally, the kit of parts further comprises one or moreconnectors, such as a T-connector, and/or an injection unit, such as aneedle. Preferably, the amount of peptides in the first file, the amountof reconstitution composition in the second file and/or the amount oroil-based adjuvant in the third file are as defined in the method forreconstituting peptides and/or the method for preparing a pharmaceuticalcomposition as defined earlier herein.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

The word “about” or “approximately” when used in association with anumerical value (e.g. about 10) preferably means that the value may bethe given value (of 10) more or less 0.1% of the value.

The sequence information as provided herein should not be so narrowlyconstrued as to require inclusion of erroneously identified bases. Theskilled person is capable of identifying such erroneously identifiedbases and knows how to correct for such errors.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show UPLC chromatograms of DP-6P (comprising SLPsrepresented by SEQ ID NOs: 1-6) in three different solvent mixtures, twohours after dissolution and storage at room temperature. (FIG. 1A) DP-6P(2.40 mg total peptide) dissolved in a mixture of 750 μL 0.1M Citricacid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μLCremophor EL; (FIG. 1B) DP-6P (2.40 mg total peptide) dissolved in 20%v/v DMSO/water; (FIG. 1C) DP-6P (2.40 mg total peptide) dissolved in 20%v/v DMSO/water with 10 mM DTT.

FIGS. 2A-2D show UPLC chromatograms of DP-6P (comprising SLPsrepresented by SEQ ID NOs: 1-6) in two different solvent mixtures and attwo time points after dissolution (t=0 and t=2h). Both solvent mixturescontain Propylene Glycol, Ethanol, water and stabilizing or reducingagents. (FIG. 2A) DP-6P (2.40 mg total peptide) dissolved in a mixtureof 600 μL water, 267 μL Propylene Glycol, 133 μL Ethanol and 1 mg/mLAscorbic acid at t=0 (FIG. 2B) DP-6P (2.40 mg total peptide) dissolvedin a mixture of 600 μL WFI, 267 μL Propylene Glycol, 133 μL Ethanol and1 mg/mL Ascorbic acid at t=2h; (FIG. 2C) DP-6P (2.40 mg total peptide)dissolved in a mixture of 750 μL 0.1M Citric acid in water, 62.5 μLPropylene Glycol, 125 μL Ethanol and 62.5 μL Cremophor EL at t=0; (FIG.2D) DP-6P (2.40 mg total peptide) dissolved in a mixture of 750 μL 0.1MCitric acid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5μL Cremophor EL at t=2h.

FIGS. 3A-3D show UPLC chromatograms of DP-6P (comprising SLPsrepresented by SEQ ID NOs: 1-6) in two different solvent mixtures. Allsolvent mixtures contain per mL 750 μL 0.1M Citric acid in water, 62.5μL Propylene Glycol, 125 μL Ethanol and either Tween20 or Cremophor EL(62.5 μL). (FIG. 3A) DP-6P (2.40 mg total peptide) dissolved in thesolvent mixture comprising Tween20 at t=0; (FIG. 3B) DP-6P (2.40 mgtotal peptide) dissolved in the solvent mixture comprising Tween20 att=2h; (FIG. 3C) DP-6P (2.40 mg total peptide) dissolved in the solventmixture comprising Cremophor EL at t=0; (FIG. 3D) DP-6P (2.40 mg totalpeptide) dissolved in the solvent mixture comprising Cremophor EL att=2h.

FIGS. 4A-4D show UPLC chromatograms of DP-6P (comprising SLPsrepresented by SEQ ID NOs: 1-6) and DP-7P (comprising SLPs representedby SEQ ID NOs: 7-13) after reconstitution and emulsification withMontanide ISA 51 VG. The solvent mixture for reconstitution contains permL 750 μL 0.1M Citric acid in water, 62.5 μL Propylene Glycol, 125 μLEthanol and Cremophor EL (62.5 μL). Prior to analysis, peptides wereextracted from the emulsion by adding an excess of the solvent mixtureand forcing phase separation by centrifugation. (FIG. 4A) DP-6P (2.4 mgtotal peptide) dissolved in 1 mL solvent mixture comprising Cremophor ELat t=0 (immediately after vaccine preparation and extraction); (FIG. 4B)DP-6P (2.4 mg total peptide) dissolved in 1 mL solvent mixturecomprising Cremophor EL at t=2h (after 2 hours storage of the vaccineproduct, followed by extraction). (FIG. 4C) DP-7P (2.8 mg total peptide)dissolved in 1 mL solvent mixture comprising Cremophor EL at t=0(immediately after vaccine preparation and extraction); (FIG. 4D) DP-7P(2.8 mg total peptide) dissolved in 1 mL solvent mixture comprisingCremophor EL at t=2h (after 2 hours storage of the vaccine product,followed by extraction).

FIG. 5 shows particle size distribution comparison (overlay) betweenDP-6P emulsions using two different citric acid concentrations. DP-6P(2.40 mg total peptide) dissolved in a mixture of 750 μL 0.05M or 0.1MCitric acid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5μL Cremophor EL, and subsequently emulsified with 1 mL Montanide ISA51VG.

FIGS. 6A-6B show particle size distribution comparison (overlay) betweenDP-6P emulsions (comprising SLPs represented by SEQ ID NOs: 1-6). DP-6P(2.40 mg total peptide) was dissolved in a mixture of 750 μL 0.1M Citricacid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μLCremophor EL and subsequently emulsified with 1 mL Montanide ISA51 VG.(FIG. 6A) three independent (repeated) preparations (prep1, prep2 andprep3) at t=0, indicating the robustness of the emulsification method.(FIG. 6B) Two independent (repeated) preparations at t=0 (prepltOh andpreplt2h) and t=2h (prep2t0h and prep2t2h), demonstrating bothrobustness of the emulsification method as well as in-use physicalstability of the emulsion for at least 2 hours at room temperature.

FIG. 7 shows a timeline for TC-1 tumor experiment.

FIGS. 8A-8D show outgrowth of TC-1 tumors in mice vaccinated with either(FIG. 8A) 40% v/v DMSO/WFI emulsified 1:1 with Montanide only (DMSO),(FIG. 8B) Reconstitution (Rec.) composition (750 μL 0.1M Citric acid inwater, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μL CremophorEL) emulsified 1:1 Montanide only (Rec. composition), or with (FIG. 8C)SLP represented by SEQ ID NO: 6 and CpG ODN1826 dissolved in 40% v/vDMSO/WFI emulsified 1:1 with Montanide (DMSO+SLP) or (FIG. 8D) SLPrepresented by SEQ ID NO: 6 and CpG ODN1826 dissolved in Reconstitution(Rec.) composition (750 μL 0.1M Citric acid in water, 62.5 μL PropyleneGlycol, 125 μL Ethanol and 62.5 μL Cremophor EL) emulsified with 1:1Montanide (Rec. composition+SLP).

FIGS. 9A-9B show Kaplan-Meier plot (survival) (FIG. 9A) and percentageof induced D^(b)-RAYNIVTF (tetramer) positive CD8⁺ T cells (FIG. 9B) ofGroup 1 mice challenged with TC-1 tumors and subsequently vaccinatedwith 40% v/v DMSO/WFI emulsified 1:1 with Montanide only (DMSO), Group 2mice challenged with TC-1 tumors and subsequently vaccinated withReconstitution composition (750 μL 0.1M Citric acid in water, 62.5 μLPropylene Glycol, 125 μL Ethanol and 62.5 μL Cremophor EL) emulsified1:1 with Montanide only (Rec. composition), Group 3 mice challenged withTC-1 tumors and subsequently vaccinated with SLP represented by SEQ IDNO: 6 and CpG ODN1826 dissolved in 40% v/v DMSO/WFI emulsified 1:1 withMontanide (DMSO+SLP) and Group 4 mice challenged with TC-1 tumors andsubsequently vaccinated with SLP represented by SEQ ID NO: 6 and CpGODN1826 dissolved in Reconstitution composition (750 μL 0.1M Citric acidin water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μL CremophorEL) emulsified 1:1 with Montanide (Rec. composition+SLP). Asteriskindicates significant difference (unpaired t-test, p=0.022); nsindicates non-significant difference (p=0.21).

FIGS. 10A-10B show UPLC chromatograms of P53 DP-5P (comprising SLPsrepresented by SEQ ID NO: 191, 193, 194, 201 and 203). P53 DP-5P wasreconstituted with a solvent mixture containing per mL 750 μL 0.1MCitric acid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol andCremophor EL (62.5 μL). Prior to analysis, the peptides were extractedfrom the final product emulsion by adding an excess of the solventmixture and forcing phase separation by centrifugation. (FIG. 10A) P53DP-5P (2.0 mg total peptide) dissolved in 1 mL solvent mixturecomprising Cremophor EL at t=0 (immediately after vaccine preparationand extraction); (FIG. 10B) P53 DP-5P (2.0 mg total peptide) dissolvedin 1 mL solvent mixture comprising Cremophor EL at t=2h (after 2 hoursstorage of the vaccine product, followed by extraction).

EXAMPLES Example 1 Introduction

The aim of this study was to find a suitable reconstitution method for amultipeptide HPV vaccine product involving dissolution of the peptideDrug Products HPV-DP-6P and HPV-DP-7P, followed by emulsification withMontanide ISA51VG. Previous studies have shown that in DMSO/WFIformulations, peptides containing one or more cysteine residues have astrong tendency to form disulfides. To improve the chemical stability ofthe Drug Products and prevent disulfide formation of the peptides, a newDMSO-free reconstitution solution was developed for reconstitution ofboth Drug Products. This new reconstitution solution should be able todissolve the Drug Product and result in a stable emulsion with MontanideISA51VG. Disulfide-formation should be minimal.

The study consists of four levels of analysis:

-   -   1. Screening for a suitable solvent combination to reconstitute        the Drug Products, monitoring dissolution of the peptides by        visual inspection.    -   2. Monitoring of the emulsion stability of the Drug Product        emulsion with Montanide. Stability is assessed by visual        inspection and by analysis of particle size of the emulsion        droplets.    -   3. Analysis of the chemical stability of the Drug Product after        reconstitution in solvents that were successful on level 1 and        2.    -   4. Analysis of the chemical stability of the Drug Product after        reconstitution and emulsification, using solvents that were        successful in level 1, 2 and 3. For this purpose, the peptides        are dissolved, emulsified with Montanide ISA 51 VG, followed by        extraction from the emulsion and analysis of the peptide        composition.

Materials

The following lyophilized peptide compositions were used: DP-5Pcomprising peptides represented herein by SEQ ID NO: 1-5 admixed atequal net weights of 0.40 mg of each peptide per vial (total amount ofprotein per vial being 2.00 mg) and 0.56 mg TFA per vial; DP-6Pcomprising peptides represented herein by SEQ ID NO: 1-6 admixed atequal net weights 0.40 mg of each peptide per vial (total amount ofprotein per vial being 2.40 mg) and 0.67 mg TFA per vial; and DP-7Pcomprising peptides represented herein by SEQ ID NO: 7-13 admixed atequal net weights of 0.40 mg of each peptide per vial (total amount ofprotein per vial being 2.80 mg) and 0.96 mg TFA per vial.

The following chemicals were used: Cremophor EL, (Sigma Aldrich,Kolliphor EL, C5135); Propylene Glycol or PG (≥99.5%, Sigma Aldrich,W294004) Ethanol or EtOH (Absolute, VWR Emprove® Ph Eur, BP, USP.Article #1.00986.1000); Citric acid or CA (≥99%, Sigma Aldrich C1909);MilliQ water (from EQP-063); Sterile Montanide ISA 51VG (SEPPIC, batch#14V011).

The following equipment was used: Syringe extrusion devices (Discofix-3T-connector, B. Braun); DMSO-resistant syringes (2 mL NORM-JECT LuerLock, Henke Sass Wolf); Waters UPLC/MS system; Malvern Mastersizer 2000;Protein Simple MFI 5200 flowcell.

Methods Dissolution

Reconstitution composition was prepared by mixing the organic andaqueous solvents before adding them to the lyophilized Drug Product. 1mL of various reconstitution compositions was added to the Drug Productand the mixture was allowed to stand for 5 minutes, while swirling thesolution several times. Physical stability was assessed by visualinspection. Chemical stability was assessed using UPLC/MS (see belowunder Chemical stability of the Drug Product solution).

Emulsification with Montanide

Solvent combinations resulting in a visually clear Drug Product solutionwere used in emulsification experiments with Montanide ISA51 VG. Unlessstated otherwise, reconstitution and emulsification was performedaccording to the protocol in Table 1. Where indicated, mixing of thecontents of syringe A and B was performed differently. These adaptationsof the procedure in Table 3 are indicated in the results section inTable 4 and Table 5.

TABLE 1 Reconstitution and emulsification of drug product (DP). StepDescription 1 At least 10 minutes and maximum 30 minutes before startformulation, thaw at room temperature 1 vial with DP, lyophilized powderfor injection. Record time of removal from the freezer (hh;min). 2Collect 1 mL reconstitution composition in a 2 mL syringe. 3 Record timeof starting the reconstitution (hh;min). 4 Add the content of thesyringe containing sterile reconstitution composition (1 mL) to the DPvial. Do not swirl the vial. Remove the syringe from the vial. 5 Allowthe mixture to stand for 2 minutes at RT, followed by gentle swirlingfor 3 minutes. If the content of the vial is not completely dissolved,vortex for 30 seconds. 6 Collect the contents of the vial (1.0 mL) in anew syringe (syringe A). 7 Collect 1.0 mL Montanide ISA 51 VG in a third2 mL syringe (syringe B). 8 Remove one of the white caps of the T-connector and firmly attach the syringe containing the peptide solutionin reconstitution composition (1.0 mL) to the connector (Syringe A). 9Remove the second white cap of the T- connector and attach the syringecontaining 1.0 mL Montanide ISA 51 (Syringe B) to the connector. 10 Turnthe switch-key and push the content of syringe A first slowly intosyringe B and then from syringe B to A. This is 1 cycle. Start thestopwatch. Repeat the cycle in total 50 times 40-50 seconds. Recordnumber of seconds (to be documented by second operator). 11 Collect thevaccine emulsion in one syringe. Remove the syringe from the T-connector and place a clean needle on the syringe.

Laser Diffraction Experiments for Testing Emulsion Stability

Emulsion stability was monitored both by visual inspection and byanalysis of the particle size distribution using a Malvern Mastersizer2000.

For particle size analysis, dilution of the emulsion was performedeither with water or with a 0.01 M citric acid in water solution toobtain the desired level of obscuration. Montanide was admixed with thereconstitution composition comprising reconstituted DP using a stirrerat a speed of 1750 rpm and a refractive index of 1.46 were applied.Particle size distribution was expressed in D(0.5) and D(0.9) for avolume-based distribution.

Micro Flow Imaging (MFI) for Testing Emulsion Stability

As a second technique for particle size analysis for assessing emulsionstability, Micro Flow Imaging was used. Prior to analysis, a dilution ofthe emulsion was prepared by adding one droplet of emulsion to 10 mL0.01M aqueous citric acid solution and mixing until homogeneous,followed by 1:100 dilution of this solution in water. Samples weremeasured in a purge volume of 0.20 mL for the duration of 0.68 minutesor per 1 million particles in one single run. The results are expressedin Equivalent Circle Diameter (ECD).

Chemical Stability of the Drug Product Solution

For samples showing complete dissolution and an emulsion stability of >2hours, the chemical stability of the Drug Product solutions (withoutadditional dilution) was monitored with UPLC/MS on a Waters Acquity UPLCsystem coupled to a Waters TQD mass spectrometer using a Waters Acquitycolumn (type: BEH130, C18, 1.7 μm, 2.1×150 mm). Data processing wasperformed with Masslynx 4.1 software. UV-detection was performed at 220nm and the mobile phase was 0.05% TFA and 1% ACN in water (buffer A) and0.05% TFA in ACN (buffer B) at a flowrate of 0.3 mL/min. The columntemperature was 65° C. and the autosampler temperature was 5° C. Aninjection volume of 5 μL was used, and the gradient profile of Table 2was applied.

UV-detection was performed during the full length of the gradient, andmass spectrometric analysis was performed from 2-30 min in the positivemode.

For analysis of chemical stability of the Drug Product solutions,samples were analyzed at various time points, at least up to 2 hoursafter dissolution.

TABLE 2 Gradient profile for UPLC/MS. Time Eluent A Eluent B (min) (%)(%) 0 87 13 0.5 87 13 5.5 79.5 20.5 17.0 68 32 22.8 45 55 28.5 45 5528.6 20 80 30.0 20 80 30.1 87 13 33.0 87 13

In-Use Chemical Stability of HPV-DP-6P and HPV-DP-7P Vaccine Emulsions

For samples showing complete dissolution, an emulsion stability of >2hours, and a chemical stability of the Drug Product solutions (withoutadditional dilution) of >2 hours, the in-use chemical stability of thevaccine emulsions with Montanide ISA 51 VG was monitored with UPLC/MS.For analysis of chemical stability of the reconstituted and emulsifiedDrug Products, samples were analyzed at various time points, at least upto 2 hours after dissolution. UPLC/MS analysis was performed accordingto the method describe above for chemical stability of the Drug Productsolution, using an extra sample preparation step for extraction of thepeptides from the vaccine emulsion. For sample preparation of emulsifiedproducts, the following steps were applied:

-   -   Take 300 μL Reconstitution Solution and add this to a 15 mL        Greiner tube    -   Add 100 μL heptane    -   Add 200 μL of the Drug Product emulsion. Pipet the solution up        and down three times.    -   Vortex the mixture for 30 seconds    -   Centrifuge the mixture for 5 minutes at 4400 rpm to obtain a        two-phase system    -   With a 20-200 μL pipette, take a 100 μL sample from the bottom        layer and transfer to a total recovery UPLC vial.    -   Analyze with UPLC/UV/MS according to the method described for        chemical stability of the Drug Product solutions.

Results Solvent Screening for Reconstitution and Emulsification

Solvents were screened to define a reconstitution composition comprisingboth an aqueous and organic fraction that is suitable for reconstitutinglyophilized peptides and forming a chemically and physically stableemulsion with Montanide. All experiments below were performed withDP-6P. The experiments were verified using DP-5P and DP-7P, but as datawere highly comparable, only the data on DP-6P are shown here. Physicalstability of the reconstituted proteins and emulsion in this screen wasassessed by visual inspection.

As organic fraction, a wide variety of organic solvents was tested. Theonly single organic solvent capable of completely dissolving DP-6P whenadmixed with WFI (water for injection) was NMP (Table 3). However, nostable emulsion with Montanide could be obtained when using NMP/WFI asreconstitution composition. The use of saline instead of WFI slightlyimproved the emulsion stability, but still no emulsions with a stabilityof ≥2h could be obtained in a reproducible manner.

TABLE 3 Solvent screening for dissolution of DP-6P. Peptides AqueousOrganic 1 solubility Emulsion stability 600 μL WFI 400 μL Particles NAGlycerol 600 μL WFI 400 μL Clear viscous NA PG solution 600 μL WFI 400μL Particles NA EtOH WFI 100-20% Particles NA DMF 800 μL WFI 200 μLClear homogeneous < 1 h NMP solution 800 μL WFI, 200 μL Clearhomogeneous < 2 h 0.9% NaCl NMP solution

Organic Solvent Mixtures in Reconstitution

No single organic solvent was identified that in combination with WFIresulted in complete dissolution of DP-6P. Therefore, combinations ofpropylene glycol and other solvents were screened as organic fraction inthe reconstitution composition. Physical stability was assessed byvisual inspection. Chemical stability was assessed using UPLC/MS.

Although still no complete dissolution of DP-6P was observed, the mostoptional solvent combination identified for dissolution of DP-6P was amixture of ethanol, propylene glycol and Cremophor EL as emulsifier withWFI (FIG. 1).

To further improve the dissolution process while limiting disulfideformation, the effect of adding several antioxidants and reducing agentsto the solvent mixture (mixture of ethanol, propylene glycol, CremophorEL and WFI) and the peptide solution was assessed. Chemical stabilitywas analyzed with UPLC/MS to monitor the extent of disulfide formation.Addition of DTT (35 molar equivalents compared to peptide) or ascorbicacid (0.1-1% solution in WFI) did not result in a reduction of disulfideformation, whereas the addition of a 0.05-0.1 M aqueous citric acidsolution to the solvent mixture resulted in both improved dissolution ofDP-6P and limited disulfide formation of area % values of ≤1% perdisulfide two hours after dissolution of the Drug Product. Citratebuffer at pH3 and a concentration of 0.05-0.1 M could not be used foremulsification because of poor peptide dissolution (data not shown).FIG. 2 presents chemical stability in time (t=0 and t=2h) of DPreconstituted in a mixture of 1 mg/mL ascorbic acid in water, propyleneglycol and ethanol versus a mixture of 0.1M citric acid in water,propylene glycol, ethanol, and Cremophor EL.

Testing Reconstitution Compositions after Reconstitution and afterSubsequent Emulsification

Cremophor EL as emulsifier is less preferred in vaccine formulationsbecause of reported side effects at higher dosages. However, thedissolving and emulsifying properties of Tween 80, cyclodextrins, andTriton X as alternatives for Cremophor EL, were inadequate (data notshown). Upon visual inspection, promising results were obtained with acombination of propylene glycol, ethanol, citric acid in WFI and 2%Tween20. The results of emulsification experiments as summarized inTable 4 show that emulsions comprising propylene glycol and ethanol incombination with either Cremophor EL or Tween 20 result in most stableemulsions. However, it appeared that the chemical stability in solutionof both DP-6P and DP-7P was significantly worse in the presence ofTween20 instead of Cremophor EL, i.e. with area % values of over 5% perdisulfide two hours after dissolution of the Drug Product (see FIG. 3for UPLC chromatograms of DP-6P; results for DP-5P and DP-7P were highlysimilar (data not shown).

Taken together, from the data presented in Table 4 and FIG. 3 it can beconcluded that Cremophor EL is preferred as an emulsifier for DP-6Pemulsions with Montanide, based on both physical and chemical stabilityof the product.

To demonstrate that the results obtained for chemical stability of DrugProduct in solution can be translated to the in-use chemical stabilityof the Drug Product in the vaccine emulsion, the in-use stability ofDP-6P and DP-7P vaccine emulsions was studied and results are presentedin FIG. 4 A, B, C and D. These results confirm that, after theemulsification step, the chemical stability of the Drug Products in thevaccine preparations is retained.

TABLE 4 Solvent screening with premixed organic and aqueous solvents (1mL). Physical Citric acid Peptides emulsion solution Organic 1 Organic 2Organic 3 solubility stability 0.1M, PG (3) EtOH (2) Cremophor + − 800μL EL (1) 0.1M, PG (1) 0 Cremophor − − 800 μL EL (1) 0.1M, PG (2) EtOH(1) Cremophor + − 800 μL EL (1) 0.1M, PG (3) EtOH (3) Cremophor − NA 800μL EL (2) 0.1M, PG (1) EtOH (1) Cremophor + − 800 μL EL (2) 0.1M, PG (2)EtOH (1) Cremophor −+ − 775 μL EL (1) 0.1M, PG (3) EtOH (2) Cremophor −+− 750 μL EL (1) 0.1M, PG (1) EtOH (2) Cremophor −+ + 750 μL EL (1) 0.1M,0 EtOH (4) Cremophor −+ − 750 μL EL (1) 0.1M, PG (3) EtOH (2) Cremophor− − 700 μL EL (1) 0.1M, PG (1) EtOH (2) Tween 20 −+ + 800 μL (1) 0.1M,PG (1) EtOH (2) Tween 20 −+ NA 750 μL (1) 0.1M, PG (1) EtOH (2) Tween 20− NA 600 μL (1) 0.1M, PG (1) EtOH (2) Tween 20, −+ + 800 μL 50% aq (1)0.1M, PG (1) EtOH (2) Tween 20, −+ + 800 μL 25% aq (1) 0.1M, PG (1) EtOH(2) Triton X − NA 750 μL (1) 0.1M, PG (1) EtOH (2) Triton X − NA 600 μL(1) 0.05M, PG (1) EtOH (1) Cremophor + − 800 μL EL (2) 0.05M, PG (2)EtOH (1) Cremophor −+ − 775 μL EL (1) 0.05M, PG (2) EtOH (1) Cremophor−+ + 750 μL EL (1) 0.05M, PG (2) EtOH (1) Cremophor − ++ 700 μL EL (1)

Fine-Tuning for Robustness in Emulsification Peptide Solubility andEmulsion Stability

A subsequent series of experiments was performed in which the ratio ofPG/EtOH/Cremophor EL was varied, two different concentrations of citricacid solution were tested, different emulsification methods were appliedand the ratio of organic vs. aqueous components of the mixture wasvaried. In general, 1 mL reconstitution composition was prepared bymixing the organic and aqueous solvents before adding them to thelyophilized Drug Product. Subsequently, an emulsion was prepared byadding 1 mL Montanide to the 1 mL of aqueous peptide solution usingdifferent mixing steps and/or connectors as indicated in Table 5 and 6.

TABLE 5 Variation in emulsification method using differentreconstitution compositions: Buffer Peptides Emulsion Emulsificationmethod composition solubility stability 20 slow* cycles and A + − 80fast* cycles 40 cycles in 40 sec A + − 20 slow cycles and B + − 80 fastcycles 10 slow cycles and B + − 40 fast cycles 20 slow cycles and C +− +80 fast cycles 10 slow cycles and C +− +− 40 fast cycles 20 slow cyclesand C +− + 40 fast cycles 40 fast cycles C +− + 40 fast cycles D +− + 20slow cycles and E − ++ 40 fast cycles 40 fast cycles E − ++ *Slowcycles: 2 seconds per cycle. Fast cycles: 1 second per cycle. A = 800 μL0.05M citric acid and 200 μL PG/EtOH/Cremophor EL (1:1:2); B = 800 μL0.05M citric acid and 200 μL PG/EtOH/Cremophor EL (2:1:1); C = 750 μL0.1M citric acid and 250 μL PG/EtOH/Cremophor EL (2:1:1); D = 750 μL0.1M citric acid and 250 μL PG/EtOH/Cremophor EL (1:2:1); and, E = 700μL 0.1M citric acid and 300 μL PG/EtOH Cremophor EL (2:1:1).

Peptide Emulsion Stability in More Detail: PSD Analysis by LaserDiffraction

For five different reconstitution compositions, the effect of differentemulsification methods on particle size was analysed with laserdiffraction, using a Malvern Mastersizer 2000. For all samples, 1 mLreconstitution composition was prepared by mixing the organic andaqueous solvents before adding them to the lyophilized Drug Product.Subsequently, an emulsion was prepared by adding 1 mL Montanide to the 1mL of aqueous peptide solution. Mixing of the organic and aqueous phaseswas performed in three different ways:

-   -   Using the T-connector process and performing mixing cycles as        indicated in Table 6;    -   Using an I-connector and performing mixing cycles as indicated        in Table 6; or,    -   Adding 1 mL Montanide to the vial containing the peptide        solution in reconstitution composition, and vortexing the        mixture during 30 seconds.        A summary of the results is presented in Table 6. Approximate        values for D(0.5) are given (volume based distribution).

TABLE 6 Emulsification of peptide formulations characteristics andaverage D(0.5) values using different reconstitution compositions:Reconstitutio Emulsification composition process Solubility D(0.5)Stability A T-connector, 20 slow − 3 μm ≥3 h and 80 fast cycles A Vortex30 seconds − 11 μm 3 h B T-connector, 20 slow +− 5-7 μm* ≥3 h and 80fast cycles B T-connector, 40 fast +− 9 μm ≥3 h cycles B I-connector, 10slow +− 11 μm ≥2 h and 40 fast cycles C T-connector, 40 fast +− 4 μm ≥2h cycles D T-connector, 40 fast +− 11 μm 1 h cycles E T-connector, 20slow + 11 μm 1 h and 80 fast cycles E I-connector, 20 slow + 12 μm 1 hand 80 fast cycles *Variation in PSD was observed for analysis dilutedin WFI or diluted in 0.01M citric acid solution A = 600 μL 0.1M citricacid and 400 μL PG/EtOH/Cremophor EL (5:4:2); B = 750 μL 0.1M citricacid and 250 μL PG/EtOH/Cremophor EL (2:1:1); C = 750 μL 0.1M citricacid and 250 μL PG/EtOH/Cremophor EL (1:2:1); D = 775 μL 0.1M citricacid and 225 μL PG/EtOH/Cremophor EL (2:1:1); and, E = 800 μL 0.1Mcitric acid and 200 μL PG/EtOH/Cremophor EL (2:1:1).

Both from Table 5 and Table 6, it appears that no difference in emulsionstability was observed between the different mixing methods and/ordifferent types of connectors used. However, vortexing the mixtureinstead of using a connector resulted in emulsions with a much largerparticle size, which is less favorable for stability. In generalemulsions with a smaller particle size are more stable.

Further, emulsion stability was improved by increasing the percentage ofthe organic fraction (mixture) in the total volume of reconstitutioncomposition. However, the highest volumes of organic content tested here(300-400 μL) resulted in decreased solubility of the Drug Product.Therefore, the optimum of organic content was between 200 and 300 μL permL (about 250 μL) reconstitution composition. In addition, variation inthe concentration of the citric acid (0.05 or 0.1M) solution did notseem to affect the emulsion stability, while slightly better dissolutionof DP-6P was obtained when a 0.1 M citric acid solution was used.

Particle Size Analysis Using Micro Flow Imaging

To study the effect of citric acid concentration on emulsion stabilityand particle size of the emulsion in more detail, additional particlesize analysis experiments were compared using the solvent that resultedin the smallest particle size after emulsification with 1 mL Montanide,i.e. a reconstitution composition with an organic- to aqueous-phaseratio of 1:3, wherein the organic phase contains PG, EtOH and CremophorEL in a ratio of PG to EtOH to Cremophor EL of 1:2:1 (Table 6). Directcomparison experiments were performed, wherein the molar amount ofcitric acid in the aqueous phase was varied (0.05 and 0.1M citric acid,i.e. an end concentration of citric acid in reconstitution compositionof 0.038 and 0.075M citric acid, respectively). DP-6P was dissolved in 1mL of such reconstitution composition, followed by emulsification with 1mL Montanide using a T-connector process and performing 50 fast mixingcycles. In these experiments, both dissolution of the Drug Product andparticle size and emulsion stability were analyzed. As a read-out, MicroFlow Imaging (MFI) was performed using an MFI 5200 in order to visualizethe particles with a camera so that irregularities can be studiedvisually. The PSD-comparison of 0.05 and 0.1M citric acid of citric acidreconstitution composition is shown in FIG. 5.

As can be seen in FIG. 5, the concentration of citric acid solution doesnot influence the PSD of the emulsion. However, dissolution of the DrugProduct was slightly better when 0.1M citric acid was used. FIG. 6presents MFI results of three independent preparations of a DP-6Pemulsion (FIG. 6, panel A) or two independent preparations analyzed attwo different time points after preparation (FIG. 6, panel B), whereinthe Drug Product has been reconstituted using the same solventcombination (750 μL 0.05M citric acid+250 μL PG/EtOH/Cremophor EL1:2:1). Very robust PSD results were obtained. In addition, theemulsions were all stable for at least 2 hours.

Application of Preferred Reconstitution Solvent and EmulsificationMethod on DP-6P and DP-7P

Since 750 μL citric acid solution+250 μL PG/EtOH/Cremophor EL 1:2:1 wasshown to give robust PSD results for DP-6P emulsions, and the use of0.1M citric acid resulted in the best dissolution of the Drug Product,this solvent combination was tested extensively for the preparation ofDP-6P and DP-7P emulsions.

DP-6P and DP-7P emulsions were prepared according to the instructions inTable 1. Briefly, 1 mL of reconstitution composition (750 μL 0.1M citricacid+250 μL PG/EtOH/Cremophor EL 1:2:1) was added to the lyophilizedDrug Product, the resulting solution was mixed with 1 mL Montanide usinga T-connector and applying 50 fast mixing cycles. PSD values for MFIanalyses are given in ECD (equivalent circle diameter) and anumber-based distribution is given. It should be noted that MFI andlaser diffraction are complementary techniques. Therefore, a directcomparison of average particle size values obtained by laser diffractionand by MFI cannot be performed.

TABLE 7 DP-6P Particle size D (0.5) values in μm. T = 0 T = 1 T = 2 T =3 Prep 1 1.77 1.71 1.70 1.71 Prep 2 1.73 1.62 1.69 1.69 Prep 3 1.61 1.731.75 1.69 Average 1.70 1.69 1.71 1.70

TABLE 8 DP-7P Particle size D (0.5) values in μm. T = 0 T = 1 T = 2 T =3 Prep 1 1.64 1.59 1.56 1.59 Prep 2 1.59 1.61 1.61 1.57 Prep 3 1.65 1.661.63 1.55 Average 1.63 1.62 1.60 1.57Table 7 and 8 show that by using a reconstitution composition comprising750 μL 0.1M citric acid+250 μL PG/EtOH/Cremophor EL 1:2:1 (i.e. 750 μL0.1M Citric acid in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and62.5 μL Cremophor EL), for both DP-6P and DP-7P emulsions can beprepared that are stable for at least 3 hours.

Example 2 Introduction

Therapeutic efficacy of SLP vaccination in combination with CpG1826 haspreviously been demonstrated in mice carrying established TC-1 tumors,which express the oncogenic E6 and E7 proteins of HPV16 (Zwaveling etal., J. Immunol. (2002) 169:350-358). To assess whether SLPs retainfunctionality in the most optimal formulation identified in Example 1(750 μL 0.1M citric acid+250 μL PG/EtOH/Cremophor EL 1:2:1), wetherapeutically vaccinated mice carrying a TC-1 tumor with an SLPharboring the D^(b)-restricted CTL epitope RAHYNIVTF (represented hereinby SEQ ID NO: 67), reconstituted either in DMSO/WFI or the novelreconstitution composition. All vaccines were subsequently emulsified inMontanide. Tumor outgrowth was monitored for 75 days. At the peak of thevaccine-induced T cell response, the percentage and phenotype ofRAHYNIVTF-specific CD8⁺ T cells was determined in the blood. SLPreconstituted in DMSO/WFI and the novel reconstitution compositionshowed a similar potency in inducing TC-1 tumor regression. Micevaccinated with the SLP reconstituted in the novel solution showed ahigher percentage of RAHYNIVTF-specific CD8⁺ T cells in the blood.

Materials

TABLE 9 Materials applied during TC-1 tumor experiment. MaterialOrigin/supplier C57BL/6 female mice, Harlan Laboratories 6-8 weeks oldMontanide ISA VG51 Seppic; batch 2384535/ U40740; exp 13 FEB. 2017 CpGODN1826 (5 mg/ml) Invivogen; cat no tlrl-1826 DMSO Mylan; lot nr 140706;exp JUNE 2017 WFI Fresenius Kabi; W005 4B03; exp 6 MAR. 2018 KLRG1-PeCy7eBioscience; cat nr 25-5893-82 CD62L-Alexa780 eBioscience; cat nr47-0621-82 CD44-Pacific Blue BioLegend; cat nr 103020 CD127-BiotineBioscience; cat nr 13-1271-85 CD8a-Alexa700 eBioscience; cat nr56-0081-82 CD3-V500 BD; cat nr 560771 Streptavidin- ThermoFischer; catnr Qdot605 Q10101MP 7-AAD viability ThermoFisher, cat nr staining A1310;exp 22 SEP. 2016 D^(b)-RAHYN1VTF Production of LUMC tetramer TrypsinGibco (Life Technologies) cat nr 25200-056 Geneticin (G418) Gibco (LifeTechnologies) cat nr 10131-027 BSA Roche Diagnostics; cat nr 10735078001Lysis buffer LUMC Pharmacy T-connector B Braun; 16494C Discofix CNORM-JECT Luer- HSW; 4010-000V0 lock 2 ml syringes NORM-JECT Luer- HSW;4010-200V0 lock 1 ml syringes BD Microlance 3; BD; cat nr 300600 25G(0.5 × 16 mm) Disposables Various; LUMC

Methods Vaccine Preparation

The following groups of mice were included in the study:

-   Group 1: (n=5) 40% v/v DMSO/WFI emulsified 1:1 with Montanide ISA    VG51.-   Group 2: (n=5) Reconstitution composition (750 μL 0.1M Citric acid    in water, 62.5 μL Propylene Glycol, 125 μL Ethanol and 62.5 μL    Cremophor EL per mL) emulsified 1:1 with Montanide ISA VG51.-   Group 3: (n=10) SLP GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR and 20 g CpG    ODN1826/mouse dissolved in 40% v/v DMSO/WFI, emulsified 1:1 with    Montanide ISA VG51.-   Group 4: (n=10) SLP GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID    NO: 6) and 20 g CpG ODN1826/mouse dissolved in Reconstitution    composition (750 μL 0.1M Citric acid in water, 62.5 μL Propylene    Glycol, 125 μL Ethanol and 62.5 μL Cremophor EL per mL), emulsified    1:1 with Montanide ISA VG51.    For mice in Group 1, a solution was prepared by admixing and    subsequently swirling 400 μL DMSO and 600 μL WFI. The solution was    taken up in a 2 mL Luer-Lock syringe (Syringe A). In another 2 mL    Luer-Lock syringe (Syringe B) 1 mL of Montanide ISA VG51 was taken    up, after which both syringes were connected to a T-connector. An    emulsion was generated by mixing the contents back and forth    extensively. After mixing, the syringes were disconnected and a 25G    needle was placed on the syringe containing the emulsion. Per mouse,    100 μL was injected in the left flank subcutaneously.    The vaccine prepared for Group 2 was prepared in an identical    manner, only differing by the use of reconstitution composition (750    μL 0.1M citric acid in water and 250 μL PG/EtOH/Cremophor EL 1:2:1,    i.e. 0.075M citric acid, 6.25% v/v propylene glycol CAS no. 57-55-6,    12.5% v/v ethanol and 6.25% v/v    polyoxyethyleneglyceroltriricinoleate 35 CAS no. 61791-12-6 in    water) instead of DMSO and WFI. The vaccine for Group 3 was prepared    by first dissolving the contents of a vial containing 1.5 mg SLP    represented herein by SEQ ID NO: 6    (GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR) in 400 μL DMSO. The SLP was    produced via Fmoc solid phase peptide synthesis (Fmoc Solid Phase    Peptide Synthesis, A Practical Approach, W. C. Chan, P. D. White    Eds, Oxford Univ. Press 2000). Then, 520 μL WFI and 80 μL CpG    ODN1826 (stock 5 mg/ml) were added to the peptide in DMSO. After    taking up this solution in a 2 mL Luer-Lock syringe, the same    vaccine preparation protocol was followed as for Group 1 by    emulsifying with Montanide ISA VG51. The preparation of vaccine for    Group 4 was identical to the protocol for Group 3, only differing in    the first step in which the contents of a vial containing 1.5 mg SLP    SEQ ID NO: 6 were dissolved in 920 μL Reconstitution composition and    adding 80 μL CpG ODN1826 (stock 5 mg/ml).

Therapeutic Vaccination

TC-1 tumor cells, expressing the oncogenic E6 and E7 proteins of HPV16were cultured in complete IMDM culture medium, supplemented with 400μg/ml geneticin. On day 0, TC-1 cells were harvested using trypsin andwashed 3 times with PBS/0.1% BSA. Directly after harvesting, 100,000TC-1 cells were injected s.c. in the right flank of 40 female C57BL/6mice. On day 8, all mice were s.c. vaccinated in the left flank asdescribed in the section Vaccine preparation. The tumor size of all micewas monitored at least twice a week using a caliper up to 75 days aftertumor challenge. The study was carried out as displayed in FIG. 7.

Measurement of Strength of T Cell Response in Blood

On day 9 after vaccination, blood was drawn from the tail vein of allmice. Blood samples were transferred to a 96-wells culture plate andcentrifuged for 5 minutes at 1600 rpm. Erythrocytes were lysed bysuspending blood cell pellets in Lysis buffer until orange colorationwas observed. Subsequently, cells were washed in FACS buffer and stainedwith the fluorescent antibodies, the D^(b)-RAHYNIVTF-APC tetramer and7-AAD mentioned in the Materials section above. After 30 minutes ofincubation on ice, cells were washed and analyzed on a BD LSRII flowcytometer in the Leiden University Medical Center (Dept. ofRheumatology).

Results Tumor Outgrowth Similar Between Vaccinated Groups

By monitoring the tumor size at least twice a week, a growth curve couldbe created for each individual mouse. In FIG. 8, the outgrowth of tumorsis shown for the different groups. Tumor regression is observed in allmice vaccinated with SLP 6 and CpG1826. Tumors in the control groupsreceiving the vehicle only, either DMSO/WFI and Montanide orReconstitution composition and Montanide, rapidly grow out. Besidesnatural variations, no clear differences are observed between bothSLP-vaccinated groups. See FIG. 9A for a Kaplan-Meier survival plot,showing no differences between the vaccinated groups.

Vaccine-Induced Tetramer-Positive CD8⁺ T Cells

FIG. 9B shows the percentage of induced D^(b)-RAYNIVTF (tetramer)positive CD8⁺ T cells. Mice in group 4 (Rec. composition+SLP) show anenhanced tetramer-positive CD8⁺ T cell response, indicating that SLP andCpG formulated in Reconstitution composition is more effective than SLPand CpG formulated in an emulsion of DMSO/WFI and Montanide in thepriming of specific murine CD8⁺ T cells. See Table 9 for the averagepercentages and standard deviations per group.

Significant differences were determined using an unpaired t-test,resulting in a p-value of p=0.022 between group 3 and 4.

Expression of KLRG1 and CD62L Indicate Favourable Antitumor ExpressionProfile after Vaccination with SLP 6

A study by Van Duikeren et al. (J Immunol, 2012; 189(7): 3397-403) aimedto identify parameters that correlated with the induction of aneffective antitumor response. By identifying such biomarkers, differentvaccine compositions can be tested in non-tumor bearing mice withprognostic value in tumor models. The authors found a correlationbetween the expression of KLRG1 and absence of CD62L expression on theone hand and effective antitumor immune responses on the other hand. Wedetermined the percentages of KLRG1- and CD62L-expressingD^(b)-RAHYNIVTF⁺ CD8⁺ T cells in the blood of vaccinated mice on day 9after vaccination using flow cytometry. No difference in percentage ofRAHYNIVTF-specific KLRG1⁺ CD62L⁻ CD8⁺ T cells is observed between groups3 and 4. Not enough RAHYNIVTF-specific CD8⁺ T cells were detected toreliably study the expression of KLRG1 and CD62L in the groups of micevaccinated with vehicle only (Group 1 and 2). See Table 10 for theaverage percentages and standard deviations per group.

TABLE 10 Average percentages and SD of tetramer⁺ CD8⁺ T cells, andaverages and percentages of expression of CD62L and KLRG1 of tetramer⁺CD8⁺ T cells in groups of mice vaccinated with SLP. % of TM+ CD8 T cells% Tm+ % CD62L− % CD62L+ Group of CD8 KLRG1+ KLRG1+ 1 Average 0.1 # # SD0 2 Average 0.1 # # SD 0.1 3 Average 1.4 51.9 4.0 SD 1.0 14.9 3.2 4Average 4.8* 52.2 4.0 SD 3.8 16.6 2.0 *indicates significant difference(p < 0.05) between groups 3 and 4 as determined by unpaired t-test.

DISCUSSION

No differences were observed in overall tumor outgrowth between thegroups of mice vaccinated with SLP 6 dissolved either in DMSO/WFI orReconstitution composition. We did observe enhanced induction ofspecific CD8⁺ T cells in the mice vaccinated with the SLP dissolved inReconstitution composition as compared to the group of mice vaccinatedwith the SLP dissolved in DMSO/WFI.

The adjuvanting properties of Montanide have been ascribed to theformation of an antigen depot and induction of local inflammation andcell death, which favors maturation of antigen-presenting cells. Theenhanced induction of tetramer⁺ CD8⁺ T cells in the group of micevaccinated with the SLP dissolved in Reconstitution composition suggeststhat the combination of this solution with Montanide constitutes anemulsion with beneficial antigen release properties or local stimulationof antigen-presenting cells. The favourable profile of KLRG1 expressionand absence of CD62L was similar between both groups of mice vaccinationwith the SLP. The data demonstrate that SLPs reconstituted in thereconstitution composition of the invention maintain their immunogeniccapacity as compared to the originally used reconstitution composition(DMSO/WFI).

Example 3 Material

The following lyophilized peptide composition was used: P53 DP5P:comprising peptides represented herein by SEQ ID NO: 191, 193, 194, 201and 203.

The following chemicals were used: Cremophor EL. (Sigma Aldrich.Kolliphor EL); Propylene Glycol (≥99.5%. Sigma Aldrich); Ethanol(Absolute. VWR Emprove® Ph Eur. BP.USP); Citric acid (≥99%. SigmaAldrich); MilliQ water; Sterile Montanide ISA 51VG (SEPPIC.)

The following equipment was used: Syringe extrusion devices (Discofix-3T-connector. B. Braun); DMSO-resistant syringes (2 mL NORM-JECT LuerLock. Henke Sass Wolf); Waters UPLC/MS system EQP-004; Protein SimpleMFI 5200

Methods

Preparation of the vaccine emulsion and a placebo emulsion was performedas described in Table 1.

Analysis of chemical stability was performed by UPLC-MS as described inExample 1, at the section describing methods for analysis of in-usechemical stability of HPV-DP-6P and HPV-DP-7P vaccine emulsionsincluding extraction of the peptides from the vaccine emulsion.

Particle size analysis was performed by Micro Flow Imaging. Prior toanalysis a dilution of the vaccine emulsion was prepared by adding 10 μLof emulsion to 10 mL Reconstitution Solution and mixing untilhomogeneous, followed by 1:500 dilution of this solution inReconstitution Solution.

Analysis settings of MFI 5200:

-   -   Method: DS500.2    -   Sample volume: 1 mL    -   Purge volume: 0.20 mL    -   Analysis: 0.68 min or 1.000.000 particles    -   Consecutive runs: 1        Results are expressed in Equivalent Circle Diameter (ECD) and a        number-based distribution is given. Particles ≥15 m are filtered        from the results since these are known to be artefacts rather        than emulsion particles.

Results Purity of Reconstituted Drug Product

Purity of the Drug Product at different time points was calculated asfollows:

Purity (%)=100%−Sum of impurities≥0.05% area

An overview of the in-use purity of the P53-DP-5P vaccine product isgiven in Error! Reference source not found.

TABLE 11 Overview of purity of reconstituted P53- DP-5P during storageat room temperature. PRODUCT: P53-DP-5P IN-USE STORAGE TIME TEST t = 0 ht = 1 h t = 2 h t = 3 h Purity 93.4 91.6 90.9 89.8 [Area %] Totalrelated 6.6 8.4 9.1 10.2 substances (≥0.05%) [Area %]As can be seen from Error! Reference source not found., purity of theDrug Products slowly decreases but is still ≥90.0% two hours aftervaccine preparation. Example chromatograms of UPLC analysis of thevaccine at t=0 and t=2h are presented in FIG. 10.Identification of main peaks and impurities with an area ≥1.0% area wasperformed using mass spectrometry. All related substances with an area≥1.0% area are reported and identified by comparing the measure m/zvalues with molecular masses of the peptide sequences and their knownand expected modifications. The resulting overview of related substancesfor an in-use storage of P53-DP-5P for up to 3 hours is given in Table12.

TABLE 12 Overview and identification of related substances ofreconstituted P53-DP-5P. In-use stability up to 3 h afterreconstitution. IN-USE STORAGE TIME SEQ RETENTION t = t = t = t = IDTIME (min.) 0 h 1 h 2 h 3 h NO Peptides and 4.97 13.00 12.02 11.87 11.63194 related 7.99 12.43 11.73 11.72 11.60 201 substances 9.57 23.90 22.3822.31 22.19 193 (≥0.05%) 14.14 30.37 28.75 28.81 28.81 191 [Area %]16.82 < 1.05 1.54 1.87 203 intramo- lecular disulfide 19.46 13.73 16.7316.21 15.56 203Recovery of Drug Product from the EmulsionThe recovery of the five individual peptides present in P53-DP-5P fromthe emulsion was verified by comparison of emulsified and non-emulsifiedsample signals. An overview of the results is given in Error! Referencesource not found.

TABLE 13 Overview of recovery by comparison of emulsified andnon-emulsified sample signals. IN-USE STORAGE TIME SEQ t = 0 h t = 1 h t= 2 h t = 3 h ID NO Recovery 97 (0.6) 97 (0.8) 96 (2.4) 94 (2.3) 194(RSD) 101 (2.0) 103 (3.8) 104 (5.1) 102 (5.1) 201 Both 98 (0.6) 99 (1.1)99 (1.9) 98 (1.3) 193 values 96 (0.3) 98 (1.4) 99 (1.7) 98 (1.1) 191given 70 (8.6) 92 (3.4) 90 (4.4) 86 (4.8) 203 as %

Physical Stability

Physical stability was analysed by particle size analysis with MFI.Results are expressed in Equivalent Circle Diameter (ECD). Mean particlesize values are given in Table 14, calculated from a number-baseddistribution.

TABLE 14 Mean particle size (ECD in μm) of P53 DP5P vaccine emulsions T= 0 h T = 1 h T = 2 h T = 3 h Prep 1 1.91 1.95 1.93 2.01 Prep 2 1.851.90 1.92 1.93 Average 1.88 1.93 1.93 1.97

Conclusion

Dissolution was successfully performed for a mixture containing 5 SLPsderived from the P53 antigen (P53 DP-5P).

Both chemical and physical in-use stability of the vaccine product wasstudied. Analysis of related substances and calculation of purity assummarized in Table 11 for P53 DP-5P shows that the purity of the DrugProduct is ≥90.0% two hours after vaccine preparation. Only one relatedsubstance with a peak area % of ≥1% was observed. MS-identificationshowed that this peak is the intramolecular disulfide of the peptide setforth in SEQ ID NO: 203.

Physical stability of the P53 DP-5P vaccine product was studied bymonitoring its particle size with MFI. The results of the particle sizeanalysis are summarized in Table 14 and show that the particle size doesnot change up to three hours after vaccine preparation. In addition, allvaccine products were monitored by visual inspection during thestability study and no phase separation was observed at any time point.

Example 4 Material and Methods

The following lyophilized peptide composition was used: PRAME DP5P:comprising peptides represented herein by SEQ ID NO: 153, 155, 156, 160and 166: A set of five PRAME-derived peptides was selected based on UPLCretention times, variation in amino acid composition, and solubility inreconstitution solution as determined by visual inspection.

Other materials and methods used were the same as in Example 3.

Results Purity of Reconstituted Drug Product

Purity of the Drug Product at different time points was calculated asfollows:

Purity (%)=100%−Sum of impurities≥0.05% area

An overview of the in-use purity of the PRAME-DP-5P vaccine product isgiven in Table 15. It should be noted that the purity of lyophilizedPRAME-DP-5P is already below 90%. Nevertheless, the very limiteddecrease in purity over time demonstrates good chemical stability ofthis reconstituted drug product.

TABLE 15 Overview of purity of reconstituted PRAME- DP-5P during storageat room temperature. PRODUCT: PRAME-DP-5P IN-USE STORAGE TIME TEST t = 0h t = 1 h t = 2 h t = 3 h Purity 82.9 83.7 82.5 82.2 [Area %] Totalrelated 17.1 16.3 17.5 17.8 substances (≥0.05%) [Area %]The low purity decrease over time indicates high chemical stability. Theimpurities with an area ≥1.0% area in PRAME-DP-5P were already presentin the mixture before reconstitution. Since no significant increase ofthese impurities was observed in this stability study, no identificationof the impurities was performed. The resulting overview of relatedsubstances for an in-use storage of PRAME-DP-5P for up to 3 hours isgiven in Table 16.

TABLE 16 Overview and identification of related substances ofreconstituted PRAME-DP-5P. In-use stability up to 3 h afterreconstitution. IN-USE STORAGE TIME SEQ RETENTION t = t = t = t = IDTIME (min.) 0 h 1 h 2 h 3 h NO Related 10.68 23.35 22.85 22.65 22.62 155substances 17.50 13.61 13.49 13.34 13.40 160 (≥0.05%) 18.91 13.49 14.1513.81 13.59 166 [Area %] 20.05 21.89 22.70 22.51 22.59 153 21.15 10.5610.52 10.17 9.98 156Recovery of Drug Product from the EmulsionThe recovery of the five individual peptides present in PRAME-DP-5P fromthe emulsion was verified by comparison of emulsified and non-emulsifiedsample signals. An overview of the results is given in Table 17.

TABLE 17 Overview of recovery by comparison of emulsified andnon-emulsified sample signals. IN-USE STORAGE TIME SEQ t = 1 h t = 1 h t= 2 h t = 3 h ID NO Recovery 81 (9.8) 87 (3.8) 84 (2.1) 81 (7.8) 155(RSD) 81 (10.4) 87 (3.7) 84 (0.9) 82 (8.2) 160 Both 74 (10.8) 84 (3.9)80 (1.0) 77 (8.1) 166 values 76 (10.7) 86 (3.3) 83 (0.2) 81 (8.6) 153given 78 (10.5) 84 (3.6) 80 (1.0) 76 (7.8) 156 as %

Conclusion

The purity of the PRAME DP-5P was not fully satisfactory (<90%), but thedecrease in purity of the reconstituted vaccine product was very limited(purity T=0 82.9%, T=3h 82.2%) confirming the benefits of thecompositions described herein.

1. (a) a first vial containing a mix of peptides that comprises orconsists of: (i) the 5 peptides comprising or consisting of thesequences selected from SEQ ID NOs: 1-5; or (ii) the 6 peptidescomprising or consisting of the sequences represented by SEQ ID NOs:1-6; or (iii) the 7 peptides comprising or consisting of the sequencesrepresented by SEQ ID NOs: 7-13; and (b) a second vial containing anoil-based adjuvant.
 2. The kit of parts according to claim 1, whereinthe oil-based adjuvant is an immune-stimulating oil-based adjuvant. 3.The kit of parts according to claim 1, wherein the mix of peptides islyophilized.
 4. The kit of parts according to claim 1, wherein thepeptides consist of a mixture of the 5 different peptides represented bySEQ ID NO: 1-5.
 5. The kit of parts according to claim 1, wherein thepeptides consist of a mixture of the 7 different peptides represented bySEQ ID NO: 7-13.
 6. The kit of parts according to claim 1, wherein thedifferent peptides in the mixture are present in substantially equalratios.
 7. The kit of parts according to claim 1, wherein the oil-basedadjuvant is selected from a mineral or non-mineral oil-based adjuvant.8. The kit of parts according to claim 7, wherein the oil-based adjuvantis selected from bio-based oil adjuvants, adjuvants based on vegetableoil or fish oil, squalene-based adjuvant, MF59, Syntex AdjuvantFormulation, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant,adjuvants based on peanut oil, Adjuvant 65, Lipovant, ASO4, Montanideadjuvants, adjuvants based on purified squalene and squalene emulsifiedwith highly purified mannide mono-oleate, and a mixture of Drakeol VRand mannide monooleate.
 9. The kit of parts according to claim 1,further comprising: (c) a third vial comprising a composition forreconstituting dried peptides, wherein the composition comprises orconsists of 60-80% v/v aqueous solution comprising an organic acid,5-10% v/v propylene glycol (CAS no. 57-55-6), 10-20% v/v lower alcohol,and 5-10% v/v non-ionic hydrophilic surfactant.
 10. The kit of partsaccording to claim 9, wherein the organic acid is citric acid.
 11. Thekit of parts according to claim 9, wherein the lower alcohol is ethanol.12. The kit of parts according to claim 9, wherein the non-ionichydrophilic surfactant: a. is a mono-, di or triglyceride, and/or b. hasa hydrophilic-lipophilic balance (HLB) value between 9 and
 14. 13. Thekit of parts according to claim 12, wherein the mono-, di ortriglyceride is an ethoxylated triglyceride.
 14. The kit of partsaccording to claim 12, wherein the non-ionic hydrophylic surfactant isethoxylated castor oil.
 15. The kit of parts according to claim 9,wherein the composition for reconstituting dried peptides comprises orconsists of 75% v/v aqueous solution comprising 0.1M citric acid, 6.25%v/v propylene glycol (CAS no. 57-55-6), 12.5% v/v ethanol, and 6.25% v/vpolyoxyethyleneglyceroltriricinoleate 35 (CAS no. 61791-12-6).
 16. Apharmaceutical composition comprising: (a) a mix of peptides thatcomprises or consists of: (i) the 5 peptides comprising or consisting ofthe sequences selected from SEQ ID NOs: 1-5; or (ii) the 6 peptidescomprising or consisting of the sequences represented by SEQ ID NOs:1-6; or (iii) the 7 peptides comprising or consisting of the sequencesrepresented by SEQ ID NOs: 7-13; and (b) an oil-based adjuvant.
 17. Thepharmaceutical composition according to claim 16, wherein the oil-basedadjuvant is an immune-stimulating oil-based adjuvant.
 18. Thepharmaceutical composition according to claim 16, wherein the oil-basedadjuvant is selected from a mineral or non-mineral oil-based adjuvant.19. The pharmaceutical composition according to claim 18, wherein theoil-based adjuvant is selected from bio-based oil adjuvants, adjuvantsbased on vegetable oil or fish oil, squalene-based adjuvant, MF59,Syntex Adjuvant Formulation, Freund's Complete Adjuvant, Freund'sIncomplete Adjuvant, adjuvants based on peanut oil, Adjuvant 65,Lipovant, ASO4, Montanide adjuvants, adjuvants based on purifiedsqualene and squalene emulsified with highly purified mannidemono-oleate, and a mixture of Drakeol VR and mannide monooleate.
 20. Thepharmaceutical composition according to claim 16, wherein thecomposition comprises or consists of 1-2 mg/mL peptides in 40-60% v/v ofthe reconstitution composition and 40-60% v/v of Montanide ISA 51VG.